Nucleic acid encoding proteins involved in protein degradation, products and methods related thereto

ABSTRACT

In accordance with the present invention, there are provided novel Siah-Mediated-Degradation-Proteins (SMDPS) and/or SCF-Complex Proteins (SCPs). Nucleic acid sequences encoding such proteins and assays employing same are also disclosed. The invention SMDPs and/or SCPs can be employed in a variety of ways, for example, for the production of anti-SMDP and/or SCP antibodies thereto, in therapeutic compositions, and methods employing such proteins and/or antibodies for drug screening, functional genomics and other applications. Also provided are transgenic non-human mammals that express the invention protein. Also provided are compositions and methods for targeting the destruction of selected polypeptides in eukaryotic cells based on the ubiquitin-independent mechanism by which ornithine decarboxylase is degraded by the 26S proteasome.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.11/441,688, filed May 25, 2006, which in turn is a continuation-in-partof U.S. patent application Ser. No. 10/679,246, filed Oct. 2, 2003,which is a continuation-in-part of U.S. patent application Ser. No.09/591,694, filed Jun. 9, 2000, now U.S. Pat. No. 6,638,734, whichclaims the benefit of U.S. Provisional Application No. 60/367,334, filedJun. 11, 1999, which was converted from U.S. Ser. No. 09/330,517, all ofwhich are incorporated herein by reference in their entirety.

Portions of the invention described herein were made in the course ofresearch supported in part by grant no. CA69381 from the NationalAcademy of Sciences from the National Institutes of Health. TheGovernment may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to nucleic acids and proteins encodedthereby.

BACKGROUND OF THE INVENTION

The temporal coordination of sequential steps within the eukaryotic cellcycle is governed in large part by protein degradation, involvingtargeted ubiquitination of specific cell cycle regulatory proteinsfollowed by their destruction by the 26S proteasome (reviewed inCiechanover, A. 1998, EMBO J., 17(24):7151-7160). Among the cell cycleregulators whose levels are controlled by ubiquitination and subsequentproteosome-dependent degradation are the cyclins (cyclins A, B, C, D1,E) and several of the cyclin-dependent kinase (cdk) inhibitory proteinsincluding p21-Waf1 and p27 Kip. Defects in this highly regulated processof protein turnover have been documented in many types of cancer.

The steps involved in polyubiquitination of specific proteins in cellsinvolve the concerted actions of E1, E2, and E3-type enzymes. E1proteins form thioester bonds in which the sulfhydryl group of internalcysteine residues binds the carboxyl amino acid of ubiquitin, therebyactivating ubiquitin for subsequent transfer to E2-family proteins. E2family proteins then transfer activated ubiquitin to the freeamino-groups of lysine side chains in target proteins directly. Moreoften, however, E2-family proteins collaborate with E3 proteins whichbind particular target proteins and orchestrate their interactions withE2s, coordinating the polyubiquitination of these target proteins inhighly regulated manners (Ciechanover, 1998, supra). E3 functions aresometimes embodied in multiprotein complexes rather than mediated by asingle protein.

The ubiquitination and degradation of a variety of cyclins,cyclin-dependent kinases (cdks) and cdk-inhibitors is temporallycontrolled during the cell cycle by SFC complexes. Theses multiproteincomplexes function as E3-like entities, and contain the Skp-1 protein,at least one Cullin-family protein, and at least one F-box protein, thusthe acronym SCF: S=Skp1; C=Cullin; F=F-box) (reviewed in Patton, E. E.et al., 1998, TIG 14(6):236-243). F-box proteins contain a conservedmotif, the F-box, which mediates their interactions with Skp-1. TheF-box proteins also contain other domains which allow them tosimultaneously bind specific substrate proteins, which are then targetedfor degradation via polyubiquitination. One such F-box proteinidentified in humans is b-Trcp, which forms a SCF complex with Skp-1 andCul-1, and which interacts with β-catenin, targeting it for degradation(Latres, et al., 1999, Oncogene, 18:849-854, and Winston, J. J. et al.,1999, Genes & Dev., 13:270-283).

Siah-family proteins represent mammalian homologs of the Drosophila Sinaprotein. Sina is required for R7 photoreceptor cell differentiationwithin the sevenless pathway. Sina binds a ubiquitin-conjugating enzyme(E2) via an N-terminal RING domain. Heterocomplexes of Sina and anotherprotein called Phyllopodia form a E3-complex which interacts with atranscriptional repressor called Tramtrack, targeting it forpolyubiqitination and proteosome-mediated degradation in the fly (Tang,A. H. et al., 1997, Cell, 90:459-467 and Li, S. et al., 1997, Cell,469-478). The destruction of Tramtrack is necessary for differentiationof R7 cells.

At present, little is known about the expression of mammalian genesrelated to the Siah-mediated-protein-degradation family of proteins innormal cells and cancers. Moreover, the diversity of functions of theSiah-mediated-protein-degradation family proteins remain unclear.Therefore, there continues to be a need in the art for the discovery ofadditional proteins that interact with theSiah-mediated-protein-degradation pathway, such as proteins that bindSiah in vivo, and especially a need for information serving tospecifically identify and characterize such proteins in terms of theiramino acid sequence. Moreover, to the extent that such molecules mightform the basis for the development of therapeutic and diagnostic agents,it is essential that the DNA encoding them be elucidated. Similarly, aneed exists to identify additional components of SCF complexes which mayoperate in concert with or independently of Siah.

A naturally occurring alternative pathway for proteasome-dependentpolypeptide degradation has been identified in ornithine decarboxylase(ODC) and antizyme (AZ) (reviewed in Coffino, Nat. Rev. Mol. Cell Biol.2:188-194, 2001). ODC directly binds to and is degraded by the 26Sproteasome through a mechanism that is catalyzed by AZ and isindependent of ubiquitin (Murakami et al., Nature 360:597-599, 1992).

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided novelisolated nucleic acids encoding a variety ofSiah-Mediated-Degradation-Proteins (SMDPs) involved in the Siah-mediatedprotein degradation pathways and/or SCF-Complex-Proteins (SCPs) involvedin SCF-mediated protein degradation pathways. Further provided arevectors containing invention nucleic acids, probes that hybridizethereto, host cells transformed therewith, antisense-nucleic acidsthereto and related compositions. The nucleic acid molecules describedherein can be incorporated into a variety of expression systems known tothose of skill in the art. In addition, the nucleic acid molecules ofthe present invention are useful as probes for assaying for the presenceand/or amount of a SMDP and/or SCP gene or mRNA transcript in a givensample. The nucleic acid molecules described herein, and oligonucleotidefragments thereof, are also useful as primers and/or templates in a PCRreaction for amplifying genes encoding SMDP and/or SCP proteins.

In accordance with the present invention, there are also providedisolated mammalian SMDP and/or SCP proteins. These proteins, orfragments thereof, are useful in bioassays, as immunogens for producinganti-SMDP and/or SCP antibodies, or in therapeutic compositionscontaining such proteins and/or antibodies. Also provided are transgenicnon-human mammals that express, or fail to express (e.g., knock-out),the invention protein.

Antibodies that are immunoreactive with invention SMDP and/or SCPproteins are also provided. These antibodies are useful in diagnosticassays to determine levels of SMDP and/or SCP proteins present in agiven sample, e.g., tissue samples, Western blots, and the like. Theantibodies can also be used to purify SMDP and/or SCP proteins fromcrude cell extracts and the like. Moreover, these antibodies areconsidered therapeutically useful to modulate the biological effect ofSMDP and/or SCP proteins in vivo.

Also provided are bioassays for identifying compounds that modulate theactivity of invention SMDP and/or SCP proteins. Methods and diagnosticsystems for determining the levels of SMDP and/or SCP protein in varioustissue samples are also provided. These diagnostic methods can be usedfor monitoring the level of therapeutically administered SMDP and/or SCPor fragments thereof to facilitate the maintenance of therapeuticallyeffective amounts. These diagnostic methods can also be used to diagnosephysiological disorders that result from abnormal levels of SMDP and/orSCP.

Also provided are systems using invention SMDPs, SCPs, or functionalfragments thereof, or other protein-degradation binding domains, fortargeting any desired protein for ubiquitination and degradation, thusenabling novel gene discovery through functional genomics strategies orproviding the basis for ablating target proteins involved in diseasesfor therapeutic purposes.

In addition, compositions and methods are provided forubiquitin-independent degradation of a target protein mediated byornithine decarboxylase (ODC). Therefore, according to one embodiment ofthe invention, an isolated polynucleotide is provided that comprises apromoter sequence operably linked to a sequence that encodes a chimericprotein that comprises (a) a target-protein binding domain operativelylinked to (b) a protein-degradation binding domain of ODC. Binding ofthe target-protein binding domain of the chimeric protein to a targetprotein in a cell induces degradation of the target protein in the cell.According to one embodiment, such a protein-degradation binding domainis an ODC C-terminal region polypeptide, including, but not limited to,a human ODC C-terminal region polypeptide. The ODC C-terminal regionpolypeptide may be a full-length ODC polypeptide or less than fulllength. According to another embodiment, the chimeric protein furthercomprises a linker sequence, for example, a flexible linker sequence,between the target-protein binding domain and the protein-degradationbinding domain of omithine decarboxylase. Examples of such flexiblelinker sequences include but are not limited to residues 31 to 94 of aBcl-2 protein, a Gly-Gly-Pro tripeptide, and [Gly-Gly-Gly-Gly-Ser]₃.Thus, for example, the chimeric protein may comprise, in order fromN-terminus to C-terminus, (a) a target-protein binding domain, (b) aflexible linker sequence, and (b) a protein-degradation binding domainof ornithine decarboxylase. According to another embodiment, thechimeric protein further comprises an epitope tag sequence. According toanother embodiment of the invention, an isolated polynucleotide isprovided that comprises: a first promoter sequence operably linked to asequence that encodes a chimeric protein that comprises a target-proteinbinding domain operatively linked to a protein-degradation bindingdomain of ODC; and a second promoter sequence operably linked to asequence that encodes an antizyme protein. Such an antizyme protein maybe, for example, a human antizyme protein, including, but not limitedto, a full-length antizyme protein. According to another embodiment ofthe invention, pharmaceutical compositions are provided that compriseone or more of the polynucleotides described above and apharmaceutically compositions further comprising one or more polyamines.According to another embodiment of the compositions of the inventioninclude, but are not limited to, cells comprising such isolatedpolynucleotides, including animals, such as mice, comprising such cells.According to another embodiment, kits are provided comprising: (a) acomposition comprising an isolated polynucleotide that comprises apromoter sequence operably linked to a sequence that encodes a chimericprotein that comprises a target-protein binding domain operativelylinked to a protein-degradation binding domain of ornithinedecarboxylase, wherein binding of the target-protein binding domain ofthe chimeric protein to a target protein in a cell induces degradationof the target protein in the cell; (b) a composition comprising anisolated polynucleotide that comprises a promoter sequence operablylinked to a sequence that encodes an antizyme protein; and (c)instructions for use.

According to another embodiment of the invention, isolated chimericproteins are provided that comprise a target-protein binding domainoperatively linked to a protein-degradation binding domain of ornithinedecarboxylase, wherein binding of the target-protein binding domain ofthe chimeric protein to a target protein in a cell induces degradationof the target protein in the cell. Such chimeric proteins may comprise alinker sequence and an epitope tag, for example, as described above.According to another embodiment of the invention, pharmaceuticalcompositions are provided that comprise one or more such chimericproteins and a pharmaceutically acceptable carrier. Such pharmaceuticalcompositions may also optionally comprise one or more polyamines.

According to another embodiment of the invention, methods are providedfor degrading a target protein in a cell. Such methods compriseproviding in a cell an isolated chimeric protein that comprises atarget-protein binding domain operatively linked to aprotein-degradation binding domain of ornithine decarboxylase, whereinbinding of the target-protein binding domain of the chimeric protein toa target protein in the cell induces degradation of the target protein.According to one embodiment of such a method, the isolated chimericpolypeptide is provided in the cell by expressing in the cell apolynucleotide that comprises a promoter sequence operably linked to asequence that encodes the chimeric protein, as described above.According to another embodiment of such a method, the isolated chimericpolypeptide and antizyme are provided in the cell by expressing in thecell (a) a first polynucleotide that comprises a first promoter sequenceoperably linked to a sequence that encodes the chimeric protein, and (b)a second polynucleotide that comprises a second promoter sequenceoperably linked to a sequence that encodes the antizyme protein.According to another embodiment of such a method, the isolated chimericprotein and the antizyme protein are provided in the cell by expressingin the cell a polynucleotide that comprises a first promoter sequenceoperably linked to a sequence that encodes the chimeric protein and asecond promoter sequence operably linked to a sequence that encodes theantizyme protein. Thus, the sequences encoding the chimeric protein andthe antizyme protein may be provided on one or more than onepolynucleotide (e.g., vector) sequences. Such methods may furthercomprise contacting the cell with a composition comprising a polyamine,including, but not limited to, spermine, spermidine, putrescine, andanalogs thereof.

According to another embodiment of the invention, method of identifyinga phenotype of a target protein in a cell are provided that comprise (a)providing in the cell an isolated chimeric protein that comprises atarget-protein binding domain operatively linked to aprotein-degradation binding domain of ornithine decarboxylase, whereinbinding of the target-protein binding domain of the chimeric protein tothe target protein in the cell induces degradation of the targetprotein, and (b) observing a change of phenotype of the cell.

According to another embodiment of the invention, methods are providedfor treating a patient having a disease characterized by expression of atarget protein in a cell of the patient, the method comprising providingin the cell an isolated chimeric protein that comprises a target-proteinbinding domain operatively linked to a protein-degradation bindingdomain of omithine decarboxylase, wherein binding of the target-proteinbinding domain of the chimeric protein to the target protein in the cellinduces degradation of the target protein.

According to another embodiment of the invention, methods are providedfor identifying a polynucleotide that encodes a target-protein bindingdomain that binds a target protein comprising: (a) expressing in thecell a vector that comprises a promoter sequence operably linked to aprotein-coding sequence that comprises (i) a test polynucleotidesequence, and (ii) a polynucleotide sequence that encodes aprotein-degradation binding domain of omithine decarboxylase, and (b)observing a change of phenotype of the cell, wherein the change ofphenotype indicates that the test polynucleotide sequence encodes thetarget-protein binding domain and that binding of the target-proteinbinding domain to the target protein in the cell induces degradation ofthe target protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an amino acid sequence comparison of SIP-L (SEQ ID NO:4)and SIP-S (SEQ ID NO:6).

FIG. 2 shows the Destruction-box amino acid consensus sequence of SAD.

FIG. 3 shows the mapping of Siah-APC interaction domains as described inExample 12.

FIG. 4 shows the results of the cell proliferation functional assay ofSIP/Siah interaction described in Example 7.

FIGS. 5A and 5B show in vitro and in vivo interaction assays of Siah-1and SIP-L as described in Examples 8 and 9, respectively.

FIGS. 6A and 6B show the mapping of SKP1, SIP-L, SAF-1 and SADinteraction domains as described in Example 13.

FIG. 7 shows the effect of Siah-1 overexpression on stability ofβ-catenin.

FIG. 8 shows a diagram of how an invention SIP communicates with theprotein ubiquitination machinery.

FIG. 9 shows a general diagram of an invention method for inducingtargeted degradation of proteins using SIP, exemplified in Example 15.

FIG. 10 shows the results of the SIP-mediated degradation of the targetTRAF6 protein, set forth in Example 15.

FIG. 11A shows a model for antizyme-dependent targeted proteindegradation by ODC-conjugated proteins. FIG. 11B shows the amino acidsequence of an ODC-fusion protein (SEQ ID NO:50).

FIG. 12 shows targeted degradation of TRAF6. HEK293T cells weretransiently transfected with plasmids encoding HA-TRAF6, HA-TRAF2, ODC,ODC-TRAF6C, ODC-RANK peptide, ODC-CD40CT or myc-antizyme in variouscombinations, as indicated. After 24 h, cell lysates were prepared andanalyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblotted using antibodies specific for hemaglutinin(HA) (TRAF6) or Myc (ODC or antizyme). The levels of TRAF6 mRNA weremeasured by Northern blot (mRNA).

FIG. 13 shows additional examples of ODC-adapter-induced degradation oftarget protein(s). FIG. 13 A shows antizyme-dependent targeteddegradation of retinoblastoma (Rb) by ODC-E7 peptide. HEK293T cells weretransiently transfected with plasmid encoding HA-Rb, ODC, ODC-E7 peptideor myc-antizyme in various combinations, as indicated. FIG. 13B showsantizyme-independent targeted degradation of Cdk2 by ODC-p21waf-1.HEK293T cells were transiently transfected with plasmid encodingmyc-Cdk2, ODC, ODC-p21waf-1 or myc-antizyme in various combinations, asindicated. FIG. 13C shows antizyme-independent targeted degradation ofIKKβ by ODC-IKKβ (leucine-zipper domain). HEK293T cells were transientlytransfected with plasmid encoding HA-IKKβ, ODC, ODC-IKKβ-LZ ormyc-antizyme in various combinations, as indicated. After 24 h, celllysates were prepared and analyzed by SDS-PAGE and immunoblotted usingantibodies specific for Rb, Cdk2, IKKβ or HSC70 (as a control).

FIG. 14 shows analysis of interactions of ODC-TRAF6C and TRAF6. HEK293Tcells were transiently transfected with plasmids encoding hemaglutinin(HA)-tagged TRAF6 and ODC, ODC-TRAF6C peptide or antizyme in variouscombinations, as indicated. After 24 h, 10 μM MG132 was added intoculture media, and the cells were incubated another 6 hours. Lysateswere subjected to immunoprecipitation using anti-myc monoclonalantibody-conjugated beads. The immunoprecipitates were analyzed bySDS-PAGE and immunoblotted using an anti-HA monoclonal antibody withECL-based detection. As a control, 0.1 volume of input cell lysate wasloaded directly in the same gel (Input).

FIG. 15 shows that targeted degradation of TRAF6 by ODC-RANK peptide isproteasome-dependent. HEK293T cells were transiently transfected withplasmid encoding HA-TRAF6, ODC-RANK peptide or myc-antizyme in variouscombinations, as indicated. After 24 h, cells were either untreated,treated with 1 μM MG132 (MG132), 1 nM Epoximycine (Epox.), 10 μMLactastacine (Lact.) or 1 μM Trypsin inhibitor (Tryp.) for 6 hours. Celllysates were prepared and analyzed by SDS-PAGE and immunoblotted usingantibodies specific for HA.

FIG. 16 shows pulse-chase analysis of ectopically expressed HA-taggedTRAF6. HEK239T cells were transiently co-transfected with plasmidsencoding HA-TRAF6 and ODC-RANK peptide, with or without myc-antizyme.After 24 hours, cells were pulse-labeled with ³⁵S-methionine andcysteine, and then chased with media lacking the labeled amino acids.Cells were lysed at the indicated times, and the expressed HA-TRAF6 wasrecovered by immunoprecipitation via a HA epitope tag.Immunoprecipitated HA-TRAF6 was subjected to SDS-PAGE and dried gelswere analyzed with a PhosphorImager. Data from pulse-chase analysis ispresented as the average±SD from duplicate experiments.

FIG. 17 shows functional analysis using ODC-E7 peptide. FIG. 17A showsdegradation of endogenous Rb protein by ODC-E7. HEK293T cells weretransiently transfected with plasmid encoding ODC, ODC-E7 peptide ormyc-Antizyme in various combinations, as indicated. After 48 h, lysateswere prepared and subjected to immunoprecipitation using anti-Rbmonoclonal antibody. The immunoprecipitates were analyzed by SDS-PAGEand immunoblotting using an anti-Rb monoclonal antibody. FIG. 17B showsthe effect of ODC-E7 on E2F reporter activity. HEK293T cells weretransiently transfected with a reporter gene plasmid that contains a E2Fresponsive element cloned upstream of a luciferase reporter gene,together with pCMVβ-gal as a transfection-efficiency control, andplasmids encoding ODC, ODC-E7 or Antizyme in various combinations, asindicated in FIG. 17A. Luciferase activity was measured in cell lysates24 hr later, and normalized relative to β-galactosidase (mean±std. dev.;n=3).

FIG. 18 shows the effect of ODC-RANK peptide and ODC-TRAF6C onIL-1-induced NFκB reporter activity. HEK293T cells were transientlytransfected with a reporter gene plasmid that contains an NFκBresponsive element cloned upstream of a luciferase reporter gene,together with pCMVβ-gal as a transfection-efficiency control, andplasmids encoding ODC, ODC-TRAF6C, ODC-RANK peptide, or Antizyme invarious combinations, as indicated. After 24 hours, cells were treatedwith 50 ng/ml IL-1 or 10 ng/ml TNFα for an additional 24 hours.Luciferase activity was measured in cell lysates and normalized relativeto β-galactosidase (mean±std. dev.; n=3).

FIG. 19 shows the requirement of FL for targeted degradation of Rb byODC-F7 peptide. HEK293T cells were transiently transfected with 0.2 μgof plasmid encoding HA-Rb (0.5 μg), ODC (0.5 μg), ODC-E7 peptide (0.5μg), ODC-E7 peptide plus FL (GGGGS) (0.5 μg), or myc-AZ (0.5 μg), invarious combinations as indicated (total DNA amount normalized). After24 hours (h), cell lysates were prepared from duplicate dishes oftransfectants, normalized for total protein content (20 μg per lane),and analyzed by SDS/PAGE/immunoblotting using antibodies specific for HA(Rb), Myc (ODC or AZ), or HSC70 (as a control) withenhanced-chemiluminescence-based detection.

FIG. 20 shows selective degradation of TRAF6 but not TRAF2 by ODCchimeric fusion polypeptides. Duplicate cultures of HEK293T cells weretransiently transfected with 0.2 μg of plasmid encoding HA-TRAF6 (0.5μg) (Upper), HA-TRAF2 (0.5 μg) (Lower), ODC (0.5 μg), ODC-C-TRAF6 (0.5μg), ODC-RANKp (0.5 μg), or myc-AZ (0.5 μg), in various combinations asindicated (total DNA amount normalized). After 24 h, cell lysates wereprepared for analysis of either polypeptide or mRNA. Immunoblot analysiswas performed by using detergent lysates normalized for total proteincontent (20 μg per lane) by SDS/PAGE/immunoblotting using antibodiesspecific for HA (TRAF6; TRAF2), Myc (ODC or AZ) withenhanced-chemiluminescence-based detection. The relative levels of TRAF6mRNA were analyzed by Northern blotting using sample normalized fortotal RNA content (10 μg per lane). Ethidium bromide staining verifiedloading of equivalent amounts of RNA for each sample.

FIG. 21 shows AZ-dependent, proteasome-dependent degradation of targetpolypeptide induced by ODC chimeric fusion polypeptide. (A)Proteasome-dependent degradation of TRAF6 by ODC-RANKp peptide. HEK293Tcells were transiently transfected with plasmids encoding HA-TRAF6 (0.5μg), ODC-RANKp (0.5 μg), or myc-AZ (0.5 μg) in various combinations, asindicated (total DNA amount normalized). After 24 h, cells were culturedwith or without 1 μM MG132, 1 nM epoximycin (Epox), 10 μM lactacystin(Lact), or 1 μM Trypsin inhibitor (Tryp) for 6 h. Cell lysates wereprepared from duplicated dishes of transfectants, normalized for totalprotein content (20 μg per lane), and analyzed bySDS/PAGE/immunoblotting using antibodies specific for HA. (B)Pulse-chase analysis of TRAF6 protein-degradation rate in ODC-RANKptransfected cells. HEK239T cells were transiently cotransfected withplasmids encoding HA-TRAF6 and ODC-RNAKp, with or without myc-AZ. After24 h, cells were pulse-labeled with [³⁵S]methionine and [³⁵S]cysteine inmethionine/cysteine-free medium, and chased with media lacking thelabeled amino acids. Cells were lysed at the indicated times, andHA-TRAF6 was recovered by immunoprecipitation by using HA antibody.Immune complexes were subjected to SDS/PAGE, and dried gels wereanalyzed by PhosphorImager (Upper). Data from pulse-chase analysis arepresented as the average (±SE) from duplicate experiments (Lower). Theblots shown are representative of duplicate experiments. (C) Pulse-chaseanalysis of IKKβ protein-degradation rate. HEK293T cells weretransiently cotransfected with plasmids encoding HA-IKKβ, with ODC orODC-IKKβ. After 24 h, cells were pulse-labeled with [³⁵S]methionine and[³⁵S]cysteine in methionine/cystein-free medium and chased with medialacking the labeled amino acids. Cells were lysed at the indicatedtimes, and HA-IKKβ was recovered by immunoprecipitation by using HAantibody. Immune complexes were subjected to SDS/PAGE, and dried gelswere analyzed by PhosphorImager (Upper). Data from pulse-chase analysisare presented as the average (±SE) from duplicate experiments (Lower).The blots shown are representative of duplicate experiments. (D) Theanalysis of Flag-Cdk2 protein-degradation rate after cycloheximidetreatment. HEK293T cells (six wells) were transiently cotransfected withplasmids encoding Flag-Cdk2 (0.5 μg), with ODC (2 μg) or ODC-p21 (2 μg).After 24 h, cells were treated with cycloheximide (50 μg/ml), lysed atthe indicated times and analyzed by SDS/PAGE/immunoblotting usingantibodies specific for Flag (Upper). Data are presented as the average(±SE) from duplicate experiments (Lower). The blots shown arerepresentative of duplicate experiments.

FIG. 22 shows the functional ablation of endogenous TRAF6 by ODC/AZsystem. HEK293T cells were transiently transfected with a reporter geneplasmid (0.1 μg) that contains a NFκB-responsive element cloned upstreamof a luciferase reporter gene, with 0.01 μg of pCMVβ-gal as atransfection-efficiency control and 0.1 μg of the indicated plasmidsencoding ODC, ODC-TRAF6C, ODC-RANKp, or AZ, in various combinations asindicated (total DNA amount normalized). After 24 h, cells were treatedwith 50 ng/ml IL-1 (Left) or 10 ng/ml TNFα (Right) for an additional 24h, and luciferase activity was measured in cell lysates and normalizedrelative to beta-galactosidase (mean±SD; n=3).

FIG. 23 shows the ablation of endogenous Rb expression by using ODC/AZsystem. (A) Scheme for E2F activation by AZ-assisted degradation of Rbpolypeptides by ODC-E7p. (B) Degradation of endogenous Rb polypeptide byODC-E7p. HEK293T cells (100-mm dish) were transiently transfected with 2μg of plasmid encoding ODC (2 μg), ODC-E7p (2 μg), or myc-AZ (2 μg) invarious combinations, as indicated (total DNA amount normalized). After48 h, lysates were normalized for total protein content and subjected toimmunoprecipitation by using 1 μg of anti-Rb monoclonal antibody. Theresulting immune complexes were analyzed by SDS/PAGE/immunoblottingusing an anti-Rb monoclonal antibody withenhanced-chemiluminescence-based detection. (C) Effect of ODC-E7p on E2Ftranscriptional activity. HEK293T cells were transiently transfectedwith a reporter gene plasmid (0.1 μg) that contains an E2F responsiveelement cloned upstream of a luciferase reporter gene, 0.01 μg ofpCMVβ-galactosidase as an transfection-efficiency control, and 0.1 μg ofthe indicated plasmids encoding ODC, ODC-E7p, or AZ, in variouscombinations as indicated (total DNA amount normalized). Luciferaseactivity was measured in cell lysates 24 h later and normalized relativeto beta-galactosidase (mean±SD; n=3).

DETAILED DESCRIPTION OF THE INVENTION

Siah-Mediated Protein Destruction by a Ubiquitin-Dependent Mechanism

In accordance with the present invention, there are provided isolatednucleic acids, which encode novel mammalianSiah-Mediated-Degradation-Proteins (SMDPs) and/or SCF-Complex-Proteins(SCPs), and functional fragments thereof. SMDPs are involved in theSiah-mediated protein degradation pathways and SCPs are involved inSCF-mediated protein degradation pathways. In some instances, these twopathways for protein degradation may operate in collaboration,particularly in cases where proteins have been identified thatphysically link SMDPs to SCPs. Invention SMDPs and/or SCPs arecontemplated herein to regulate protein degradation, either byactivating or inhibiting such protein degradation.

As used herein, invention SMDPs are proteins that participate in theSiah-mediated protein degradation pathway. The term “Siah” refers to themammalian family of proteins encoded by at least two genes referred toas SIAH1 and SIAH2 (Hu, G. et al., 1997, Genomics 46:103-111). Liketheir Drosophila counterpart protein Sina, the Siah-1 and Siah-2proteins bind ubiquitin conjugating enzymes (UBCs) via an N-terminalRING domain and target other proteins for degradation.

As used herein, invention SCPs are proteins that participate in theSkp-1, Cullin, F-box (SCF) protein degradation pathway. These proteinscan be components of SCF complexes or proteins that associate with SCFcomplexes. In some instances an invention protein may fulfill therequirements of both a SMDP and a SCP.

Using yeast two-hybrid screening methods, targets of Siah-mediatedprotein degradation have been identified demonstrating the involvementof Siah and other inventions SMDPs and/or SCPs in pathways involved incell growth regulation in cancers. For example, evidence is providedherein demonstrating that Siah-1 interacts indirectly with SCF complexesthrough associations with an invention SIP protein. Siah-1 has also beenfound to be an important regulator of cell growth, through its effectson ubiquitination and degradation of β-catenin and possibly other targetproteins including an invention protein SAD.

Thus far, the only reported target of Siah-mediated degradation is DCC(Hu, G. et al., 1997, Genes & Dev., 11:2701-2714), a putative tumorsuppressor protein encoded by a gene which is commonly disrupted incolon cancers (Fearon, E. R. et al., 1990, Science, 247:49-56). Theinvolvement of DCC in colon cancers however has recently beenquestioned, and it appears that a different gene located near DCC on18q21 is the primary target of deletions in this chromosomal region(Fearon, E. R. et al., 1990, supra). However, the DCC protein hasrecently been shown to deliver either pro-apoptotic or anti-apoptoticsignals, depending on whether it is complexed with its ligand Netrin.These observations suggest that deletion or inactivation of DCC couldpotentially contribute to tumorigensis by removing a pro-apoptoticinfluence from cells.

Another connection between the Siah-family of proteins and tumorsuppressor genes has been found for p53. For example, the Siah-1 gene ofmice was found among a group of immediate-early genes induced by p53using a hemopoietic cell line as a model for p53-induced cell cyclearrest and apoptosis (Amson, R. B. et al., 1996, PNAS, USA,93:3953-3957). Expression of Siah-1 was also indirectly correlated withincreased apoptosis in tumor xenograph experiments, suggesting thatSiah-1 could function as a tumor suppressor in some contexts (Nemani, M.et al., 1996, Proc. Natl. Sci. USA 93:9039-9042).

It has also been found that Siah-1 over-expression can induce cell cyclearrest independently of apoptosis in epithelial cancer cells (Matsuzawa,et al., 1998, EMBO J., 17(10):2736-2747). Moreover, UV-irradiation atsubapoptotic doses was shown to induce Siah-1 gene expression in MCF7breast cancer cells and to promote cell cycle arrest. These and otherdata have implicated Siah-1 in a p53-inducible pathway for cell cyclearrest which runs parallel to the well-studied p21-Waf1 pathway(Matsuzawa, et al. 1998, supra).

The phrase “SMDP and/or SCP” refers to substantially pure native SMDPand/or SCP, or recombinantly produced proteins, including naturallyoccurring allelic variants thereof encoded by mRNA generated byalternative splicing of a primary transcript, and further includingfragments thereof which retain at least one native biological activity,such as immunogenicity, the ability to bind to a SMDP and/or SCP, andthe like. Exemplary SMDPs and/or SCPs referred to herein include aminoacid sequences set forth in SEQ ID Nos:2 (Siah-1α), 4 (SIP-L), 6(SIP-S), 8 (SAF-1α), 10 (SAF-1β), 12 (SAF-2) and 14 (SAD). Inventionisolated SMDPs and/or SCPs are substantially pure and free of cellularcomponents and/or contaminants normally associated with a native in vivoenvironment.

As used herein, the term “Siah-1α” refers to a splice-variant member ofthe mammalian, preferably human, Siah-family of proteins. The inventionSiah-1α protein, or functional fragment thereof, is characterized byhaving the ability to bind to at least one or more of the proteinsselected from APC (Kinzler K. W., et al., 1996, Cell, 87(2):159-170);BAG-1 (Takayama et al., 1995, Cell, 80(2):279-284); SIP-L; SIP-S; orother Siah proteins, such as Siah-1. Thus, homodimers of Siah-1α arecontemplated herin. Invention Siah-1α proteins differ from Siah-1β (setforth as SIAH-1 in Hu et al., 1997, Genomics, 46:103-111) by containingan additional 16 amino acids at the amino-terminus. Thus, preferredinvention Siah-1α proteins, and fragments thereof, comprise at least aportion of the 16 N-terminal amino acids of SEQ ID NO:2. A particularlypreferred Siah-1α protein is set forth in SEQ ID NO:2.

In accordance with another embodiment of the invention, Siah-1 has beenfound to interact with an invention protein referred to herein as the“SIP” family. As used herein, the term “SIP” refers to any species,preferably mammalian, more preferably human, Siah-1-Interacting Protein(SIP). The invention SIP proteins, or functional fragments thereof, arecharacterized by having the ability to bind to at least one or more ofthe proteins selected from Siah-1, Skp1, or other SIP proteins. Thus,homodimers of invention SIP proteins are contemplated herein. The SIPgene has been found to encode at least two proteins through alternativemRNA splicing: SIP-L (L, for long; SEQ ID NO:4), and SIP-S (S, forshort; SEQ ID NO:6). A sequence comparison of SIP-L to SIP-S is setforth in FIG. 1. To further identify potential targets ofSiah-1-mediated ubiquitin/proteasome protein degradation, yeasttwo-hybrid screens of cDNA libraries were performed using the inventionhuman SIP-L protein (SEQ ID NO:4) as a bait. Such screen resulted in theidentification of Skp1 (Zhang et al., 1995, Cell, 82(6):915-925).

As shown in FIG. 8, an invention SIP protein binds simultaneously toSiah and Skp, proteins known to bind directly or indirectly,respectively, to ubiquitin-conjugating enzymes (E2s). Therefore, inaccordance with the present invention, this characteristic of SIP isuseful for methods for targeting desired proteins for degradation, via aubiquitin/proteosome-dependent mechanism (see, e.g., Example 15).

In accordance with another embodiment of the invention, using a yeasttwo-hybrid screen with Skp1 as bait (set forth in the Examples), twoadditional invention SMDP and/or SCP proteins were identified asSkp1-interacting proteins, which are referred to herein as SAF-1 andSAD. As used herein the term “SAF-1” refers to Skp 1-Associated F-boxprotein-1. The invention SAF-1 proteins, or functional fragmentsthereof, are characterized by having the ability to bind to at least oneor more of the proteins selected from Skp1, SIP, such as SIP-L, or SAD.Invention SAF-1 proteins are further characterized as containing an“F-box” amino acid domain. Exemplary SAF-1 proteins include SAF-1α (SEQID NO:8) and SAF-1β (SEQ ID NO:10). An exemplary F-box domain is setforth as amino acids 256-296 of SEQ ID NO:8 and amino acids 335-375 ofSEQ ID NO:10 (see FIG. 6). SAF-1 beta has the same F-Box as alfa, thelocation is in amino acids 335-375. In accordance with the presentinvention, a homologue of SAF-1 protein has also been identified in theNCBI BLAST data base (Human DNA sequence from clone 341E18 on chromosome6p11.2-12.3, AL031178) which shares significant homology with F-boxdomain of SAF-1. The invention homolog is referred to herein as SAF-2and is set forth in SEQ ID Nos: 11 and 12.

As used herein the term “SAD” refers to Skp1-Associated Destruction-boxprotein. The invention SAF-1 proteins, or functional fragments thereof,are characterized by having the ability to bind to at least one or moreof the proteins selected from Skp1, SIP, such as SIP-L, or SAF-1. It isalso contemplated herein that SAD has the ability to bind to SAF-2.Invention SAD proteins are further characterized as containing an“D-box” (Destruction-box) amino acid domain. An exemplary SAD protein isset forth herein as SEQ ID NO: 14. As used herein, the D-box domaincomprises the consensus amino acid sequence -DSGX₁X₂S-, wherein X₁ ispreferably selected from a hydrophobic amino acid, such as Y, I, L, M,F, W, or V; and X₂ is any amino acid (see FIG. 2). A preferred D-boxdomain comprises the sequence set forth as amino acids 144-149 of SEQ IDNO:14.

Thus, exemplary functional fragments of an invention SAD proteincomprise at least amino acids 144-149 of SEQ ID NO: 14. Alsocontemplated herein are functional fragments of inventions SAD proteinsthat bind to a SIP protein, and preferably comprise at least amino acids360-447 of SEQ ID NO:14. Functional fragments of inventions SAD proteinsthat bind to a Skp1 protein preferably comprise at least amino acids128-359 of SEQ ID NO: 14. Functional fragments of inventions SADproteins that bind to a SAF-1 protein preferably comprise at least aminoacids 1-127 of SEQ ID NO: 14.

The nucleic acid molecules described herein are useful for producinginvention proteins, when such nucleic acids are incorporated into avariety of protein expression systems known to those of skill in theart. In addition, such nucleic acid molecules or fragments thereof canbe labeled with a readily detectable substituent and used ashybridization probes for assaying for the presence and/or amount of aninvention SMDP and/or SCP gene or mRNA transcript in a given sample. Thenucleic acid molecules described herein, and fragments thereof, are alsouseful as primers and/or templates in a PCR reaction for amplifyinggenes encoding invention proteins described herein.

The term “nucleic acid” (also referred to as polynucleotides)encompasses ribonucleic acid (RNA) or deoxyribonucleic acid (DNA),probes, oligonucleotides, and primers. DNA can be either complementaryDNA (cDNA) or genomic DNA, e.g. a gene encoding a SMDP and/or SCP. Onemeans of isolating a nucleic acid encoding an SMDP and/or SCPpolypeptide is to probe a mammalian genomic library with a natural orartificially designed DNA probe using methods well known in the art. DNAprobes derived from the SMDP and/or SCP gene are particularly useful forthis purpose. DNA and cDNA molecules that encode SMDP and/or SCPpolypeptides can be used to obtain complementary genomic DNA, cDNA orRNA from mammalian (e.g., human, mouse, rat, rabbit, pig, and the like),or other animal sources, or to isolate related cDNA or genomic clones bythe screening of cDNA or genomic libraries, by methods described in moredetail below. Examples of nucleic acids are RNA, cDNA, or isolatedgenomic DNA encoding an SMDP and/or SCP polypeptide. Such nucleic acidsmay include, but are not limited to, nucleic acids comprisingsubstantially the same nucleotide sequence as set forth in SEQ ID Nos: 1(Siah-1α), 3 (SIP-L), 5 (SIP-S), 7 (SAF-1α), 9 (SAF-1β), 11 (SAF-2) and13 (SAD).

Use of the terms “isolated” and/or “purified” in the presentspecification and claims as a modifier of DNA, RNA, polypeptides orproteins means that the DNA, RNA, polypeptides or proteins so designatedhave been produced in such form by the hand of man, and thus areseparated from their native in vivo cellular environment, and aresubstantially free of any other species of nucleic acid or protein. As aresult of this human intervention, the recombinant DNAs, RNAs,polypeptides and proteins of the invention are useful in ways describedherein that the DNAs, RNAs, polypeptides or proteins as they naturallyoccur are not.

Invention proteins can be obtained from any species of organism, such asprokaryotes, eukaryotes, plants, fungi, vertebrates, invertebrates, andthe like. A particular species can be d mammalian, As used herein,“mammalian” refers to a subset of species from which an invention SMDPand/or SCP is derived, e.g., human, rat, mouse, rabbit, monkey, baboon,bovine, porcine, ovine, canine, feline, and the like. A preferred SMDPand/or SCP herein, is human SMDP and/or SCP.

In one embodiment of the present invention, cDNAs encoding the inventionSMDPs and/or SCPs disclosed herein comprise substantially the samenucleotide sequence as the coding region set forth in any of SEQ IDNOs:1, 3, 5, 7, 9, 11 and 13. Preferred cDNA molecules encoding theinvention proteins comprise the same nucleotide sequence as as thecoding region set forth in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11 and 13.

As employed herein, the term “substantially the same nucleotidesequence” refers to DNA having sufficient identity to the referencepolynucleotide, such that it will hybridize to the reference nucleotideunder moderately stringent hybridization conditions. In one embodiment,DNA having substantially the same nucleotide sequence as the referencenucleotide sequence encodes substantially the same amino acid sequenceas that set forth in any of SEQ ID Nos:2, 4, 6, 8, 10, 12 or 14. Inanother embodiment, DNA having “substantially the same nucleotidesequence” as the reference nucleotide sequence has at least 60% identitywith respect to the reference nucleotide sequence. DNA having at least70%, more preferably at least 90%, yet more preferably at least 95%,identity to the reference nucleotide sequence is preferred.

This invention also encompasses nucleic acids which differ from thenucleic acids shown in SEQ ID NOs:1, 3, 5, 7, 9, 11 and 13, but whichhave the same phenotype. Phenotypically similar nucleic acids are alsoreferred to as “functionally equivalent nucleic acids”. As used herein,the phrase “functionally equivalent nucleic acids” encompasses nucleicacids characterized by slight and non-consequential sequence variationsthat will function in substantially the same manner to produce the sameprotein product(s) as the nucleic acids disclosed herein. In particular,functionally equivalent nucleic acids encode polypeptides that are thesame as those encoded by the nucleic acids disclosed herein or that haveconservative amino acid variations. For example, conservative variationsinclude substitution of a non-polar residue with another non-polarresidue, or substitution of a charged residue with a similarly chargedresidue. These variations include those recognized by skilled artisansas those that do not substantially alter the tertiary structure of theprotein.

Further provided are nucleic acids encoding SMDP and/or SCP polypeptidesthat, by virtue of the degeneracy of the genetic code, do notnecessarily hybridize to the invention nucleic acids under specifiedhybridization conditions. Preferred nucleic acids encoding the inventionSMDPs and/or SCPs are comprised of nucleotides that encode substantiallythe same amino acid sequence as set forth in SEQ ID Nos:2, 4, 6, 8, 10,12 or 14.

Thus, an exemplary nucleic acid encoding an invention SMDP and/or SCPmay be selected from: (a) DNA encoding the amino acid sequence set forthin SEQ ID Nos:2, 4, 6, 8, 10, 12 or 14, (b) DNA that hybridizes to theDNA of (a) under moderately stringent conditions, wherein said DNAencodes biologically active SMDP and/or SCP, or (c) DNA degenerate withrespect to either (a) or (b) above, wherein said DNA encodesbiologically active SMDP and/or SCP.

Hybridization refers to the binding of complementary strands of nucleicacid (i.e., sense:antisense strands or probe:target-DNA) to each otherthrough hydrogen bonds, similar to the bonds that naturally occur inchromosomal DNA. Stringency levels used to hybridize a given probe withtarget-DNA can be readily varied by those of skill in the art.

The phrase “stringent hybridization” is used herein to refer toconditions under which polynucleic acid hybrids are stable. As known tothose of skill in the art, the stability of hybrids is reflected in themelting temperature (T_(m)) of the hybrids. In general, the stability ofa hybrid is a function of sodium ion concentration and temperature.Typically, the hybridization reaction is performed under conditions oflower stringency, followed by washes of varying, but higher, stringency.Reference to hybridization stringency relates to such washingconditions.

As used herein, the phrase “moderately stringent hybridization” refersto conditions that permit target-DNA to bind a complementary nucleicacid that has about 60% identity, preferably about 75% identity, morepreferably about 85% identity to the target DNA; with greater than about90% identity to target-DNA being especially preferred. Preferably,moderately stringent conditions are conditions equivalent tohybridization in 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDSat 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 65° C.

The phrase “high stringency hybridization” refers to conditions thatpermit hybridization of only those nucleic acid sequences that formstable hybrids in 0.018M NaCl at 65° C. (i.e., if a hybrid is not stablein 0.018M NaCl at 65° C., it will not be stable under high stringencyconditions, as contemplated herein). High stringency conditions can beprovided, for example, by hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C.

The phrase “low stringency hybridization” refers to conditionsequivalent to hybridization in 10% formamide, 5×Denhart's solution,6×SSPE, 0.2% SDS at 42° C., followed by washing in 1×SSPE, 0.2% SDS, at50° C. Denhart's solution and SSPE (see, e.g., Sambrook et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989) are well known to those of skill in the art as are othersuitable hybridization buffers.

As used herein, the term “degenerate” refers to codons that differ in atleast one nucleotide from a reference nucleic acid, e.g., SEQ ID NOs: 1,3, 5, 7, 9, 11 and 13, but encode the same amino acids as the referencenucleic acid. For example, codons specified by the triplets “UCU”,“UCC”, “UCA”, and “UCG” are degenerate with respect to each other sinceall four of these codons encode the amino acid serine.

Preferred nucleic acids encoding the invention polypeptide(s) hybridizeunder moderately stringent, preferably high stringency, conditions tosubstantially the entire sequence, or substantial portions (i.e.,typically at least 15-30 nucleotides) of the nucleic acid sequence setforth in SEQ ID NOs: 1, 3, 5, 7, 9, 11 and 13.

The invention nucleic acids can be produced by a variety of methodswell-known in the art, e.g., the methods described herein, employing PCRamplification using oligonucleotide primers from various regions of SEQID NOs: 1, 3, 5, 7, 9, 11 and 13, and the like.

In accordance with a further embodiment of the present invention,optionally labeled SMDP and/or SCP-encoding cDNAs, or fragments thereof,can be employed to probe library(ies) (e.g., cDNA, genomic, and thelike) for additional nucleic acid sequences encoding novel mammalianSMDPs and/or SCPs. Construction of suitable mammalian cDNA libraries iswell-known in the art. Screening of such a cDNA library is initiallycarried out under low-stringency conditions, which comprise atemperature of less than about 42° C., a formamide concentration of lessthan about 50%, and a moderate to low salt concentration.

Presently preferred probe-based screening conditions comprise atemperature of about 37° C., a formamide concentration of about 20%, anda salt concentration of about 5×standard saline citrate (SSC; 20×SSCcontains 3M sodium chloride, 0.3M sodium citrate, pH 7.0). Suchconditions will allow the identification of sequences which have asubstantial degree of similarity with the probe sequence, withoutrequiring perfect homology. The phrase “substantial similarity” refersto sequences which share at least 50% homology. Preferably,hybridization conditions will be selected which allow the identificationof sequences having at least 70% homology with the probe, whilediscriminating against sequences which have a lower degree of homologywith the probe. As a result, nucleic acids having substantially the samenucleotide sequence as SEQ ID NOs: 1, 3, 5, 7, 9, 11 and 13 areobtained.

As used herein, a nucleic acid “probe” is single-stranded DNA or RNA, oranalogs thereof, that has a sequence of nucleotides that includes atleast 14, at least 20, at least 50, at least 100, at least 200, at least300, at least 400, or at least 500 contiguous bases that are the same as(or the complement of) any contiguous bases set forth in any of SEQ IDNOs:1, 3, 5, 7, 9, 11 and 13. Preferred regions from which to constructprobes include 5′ and/or 3′ coding regions of SEQ ID NOs:1, 3, 5, 7, 9,11 and 13. In addition, the entire cDNA encoding region of an inventionSMDP and/or SCP, or the entire sequence corresponding to SEQ ID NOs:1,3, 5, 7, 9, 11 and 13, may be used as a probe. Probes may be labeled bymethods well-known in the art, as described hereinafter, and used invarious diagnostic kits.

It is understood that a SMDP and/or SCP-encoding nucleic acid moleculeof the invention, as used herein, specifically excludes previously knownnucleic acid molecules consisting of nucleotide sequences havingidentity with the SMDP and/or SCP-encoding nucleotide sequence (e.g.,SEQ ID NO:NOs:1, 3, 5, 7, 9, 11, 13), such as Expressed Sequence Tags(ESTs), Sequence Tagged Sites (STSs) and genomic fragments, deposited inpublic databases such as the nr, dbest, dbsts, gss and htgs databases,which are available for searching athttp://www.ncbi.nlm.nih.gov/blast/blast.cgi?Jform=0, using the programBLASTN 2.0.9 described by Altschul et al., Nucleic Acids Res.25:3389-3402 (1997).

In particular, a SMDP and/or SCP-encoding nucleic acid moleculespecifically excludes nucleic acid molecules consisting of any of thenucleotide sequences having the Genbank (gb), EMBL (emb) or DDBJ (dbj)accession numbers described below. Similarly, a SMDP and/or SCPpolypeptide fragment specifically excludes the amino acid fragmentsencoded by the nucleotide sequences having the GenBank accession numbersdescribed below. GenBank accession numbers specifically excluded includeNCBI ID: AA054272, AA258606, AA923663, AA418482, and A1167464. The humansequence referenced as GenBank accession No. AL031178 is alsospecifically excluded from an invention SMDP and/or SCP-encoding nucleicacid.

As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal. Any label or indicating means can be linked to invention nucleicacid probes, expressed proteins, polypeptide fragments, or antibodymolecules. These atoms or molecules can be used alone or in conjunctionwith additional reagents. Such labels are themselves well-known inclinical diagnostic chemistry.

The labeling means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturation to form afluorochrome (dye) that is a useful immunofluorescent tracer. Adescription of immunofluorescent analytic techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

In one embodiment, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, and the like. In anotherembodiment, radioactive elements are employed labeling agents. Thelinking of a label to a substrate, i.e., labeling of nucleic acidprobes, antibodies, polypeptides, and proteins, is well known in theart. For instance, an invention antibody can be labeled by metabolicincorporation of radiolabeled amino acids provided in the culturemedium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981).Conventional means of protein conjugation or coupling by activatedfunctional groups are particularly applicable. See, for example,Aurameas et al., Scand. J. Immunol., Vol. 8, Suppl. 7:7-23 (1978),Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.

In accordance with another embodiment of the present invention, thereare provided isolated mammalian Siah-Mediated-Degradation-Proteins(SMDPS) and/or SCF-Complex-Proteins (SCPs), and fragments thereofencoded by invention nucleic acid. The phrase “SMDP and/or SCP” refersto substantially pure native SMDP and/or SCP, or recombinantly producedproteins, including naturally occurring allelic variants thereof encodedby mRNA generated by alternative splicing of a primary transcript, andfurther including fragments thereof which retain at least one nativebiological activity, such as immunogenicity, the ability to bind toanother member of the SMDP and/or SCP families, or to homodimerize. Inanother embodiment, SMDPs and/or SCPs referred to herein, are thosepolypeptides specifically recognized by an antibody that alsospecifically recognizes a SMDP and/or SCP (preferably human) includingan amino acid sequence set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12 and14. Invention isolated SMDPs and/or SCPs are substantially pure and freeof cellular components and/or contaminants normally associated with anative in vivo environment.

Presently preferred SMDPs and/or SCPs of the invention include proteinsthat comprise substantially the same amino acid sequences as the proteinsequence set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 14, as well asbiologically active, functional fragments thereof.

Those of skill in the art will recognize that numerous residues of theabove-described sequences can be substituted with other, chemically,sterically and/or electronically similar residues without substantiallyaltering the biological activity of the resulting receptor species. Inaddition, larger polypeptide sequences containing substantially the samesequence as amino acids set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12 and14 therein (e.g., splice variants) are contemplated.

As employed herein, the term “substantially the same amino acidsequence” refers to amino acid sequences having at least about 70%identity with respect to the reference amino acid sequence, andretaining comparable functional and biological activity characteristicof the protein defined by the reference amino acid sequence. Preferably,proteins having “substantially the same amino acid sequence” will haveat least about 80%, more preferably 90% amino acid identity with respectto the reference amino acid sequence; with greater than about 95% aminoacid sequence identity being especially preferred. It is recognized,however, that polypeptides (or nucleic acids referred to hereinbefore)containing less than the described levels of sequence identity arisingas splice variants or that are modified by conservative amino acidsubstitutions, or by substitution of degenerate codons are alsoencompassed within the scope of the present invention.

The term “biologically active” or “functional”, when used herein as amodifier of invention SMDP and/or SCP(s), or polypeptide fragmentsthereof, refers to a polypeptide that exhibits functionalcharacteristics similar to SMDP and/or SCP. For example, one biologicalactivity of SMDP and/or SCP is the ability to bind, preferably in vivo,to at least one other member of the SMDP and/or SCP families ofproteins, or to homodimerize, or to mediate protein degradation via anSFC complex as described herein. Such SMDP and/or SCP binding activitycan be assayed, for example, using the methods described herein. Anotherbiological activity of SMDP and/or SCP is the ability to act as animmunogen for the production of polyclonal and monoclonal antibodiesthat bind specifically to an invention SMDP and/or SCP. Thus, aninvention nucleic acid encoding SMDP and/or SCP will encode apolypeptide specifically recognized by an antibody that alsospecifically recognizes the SMDP and/or SCP protein (preferably human)including the amino acid set forth in SEQ ID NOs:2, 4, 6, 8, 10, 12 and14. Such immunologic activity may be assayed by any method known tothose of skill in the art. For example, a test-polypeptide encoded by aSMDP and/or SCP cDNA can be used to produce antibodies, which are thenassayed for their ability to bind to an invention SMDP and/or SCPprotein including the sequence set forth in SEQ ID Nos:2, 4, 6, 8, 10,12 or 14. If the antibody binds to the test-polypeptide and the proteinincluding the sequence encoded by SEQ ID NOs:2, 4, 6, 8, 10, 12 or 14with substantially the same affinity, then the polypeptide possesses therequisite immunologic biological activity.

The invention SMDPs and/or SCPs can be isolated by a variety of methodswell-known in the art, e.g., recombinant expression systems describedherein, precipitation, gel filtration, ion-exchange, reverse-phase andaffinity chromatography, and the like. Other well-known methods aredescribed in Deutscher et al., Guide to Protein Purification: Methods inEnzymology Vol. 182 (Academic Press, (1990)), which is incorporatedherein by reference. Alternatively, the isolated polypeptides of thepresent invention can be obtained using well-known recombinant methodsas described, for example, in Sambrook et al., supra., 1989).

An example of the means for preparing the invention polypeptide(s) is toexpress nucleic acids encoding the SMDP and/or SCP in a suitable hostcell, such as a bacterial cell, a yeast cell, an amphibian cell (i.e.,oocyte), or a mammalian cell, using methods well known in the art, andrecovering the expressed polypeptide, again using well-known methods.Invention polypeptides can be isolated directly from cells that havebeen transformed with expression vectors as described below herein. Theinvention polypeptide, biologically functional fragments, and functionalequivalents thereof can also be produced by chemical synthesis. Forexample, synthetic polypeptides can be produced using AppliedBiosystems, Inc. Model 430A or 431A automatic peptide synthesizer(Foster City, Calif.) employing the chemistry provided by themanufacturer.

Also encompassed by the term SMDP and/or SCP are functional fragments orpolypeptide analogs thereof. The term “functional fragment” refers to apeptide fragment that is a portion of a full length SMDP and/or SCPprotein, provided that the portion has a biological activity, as definedabove, that is characteristic of the corresponding full length protein.For example, a functional fragment of an invention SMDP and/or SCPprotein can have the protein:protein binding activity prevalent in SMDPsand/or SCPs. In addition, the characteristic of a functional fragment ofinvention SMDP and/or SCP proteins to elicit an immune response isuseful for obtaining an anti-SMDP and/or SCP antibodies. Thus, theinvention also provides functional fragments of invention SMDP and/orSCP proteins, which can be identified using the binding and routinemethods, such as bioassays described herein.

The term “polypeptide analog” includes any polypeptide having an aminoacid residue sequence substantially the same as a sequence specificallyshown herein in which one or more residues have been conservativelysubstituted with a functionally similar residue and which displays theability to functionally mimic an SMDP and/or SCP as described herein.Examples of conservative substitutions include the substitution of onenon-polar (hydrophobic) residue such as isoleucine, valine, leucine ormethionine for another, the substitution of one polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, between glycine and serine, the substitutionof one basic residue such as lysine, arginine or histidine for another,or the substitution of one acidic residue, such as aspartic acid orglutamic acid for another.

The amino acid length of functional fragments or polypeptide anlogs ofthe present invention can range from about 5 amino acids up to thefull-length protein sequence of an invention SMDP and/or SCP. In certainembodiments, the amino acid lengths include, for example, at least about10 amino acids, at least about 20, at least about 30, at least about 40,at least about 50, at least about 75, at least about 100, at least about150, at least about 200, at least about 250 or more amino acids inlength up to the full-length SMDP and/or SCP protein sequence.

As used herein the phrase “conservative substitution” also includes theuse of a chemically derivatized residue in place of a non-derivatizedresidue, provided that such polypeptide displays the required bindingactivity. The phrase “chemical derivative” refers to a subjectpolypeptide having one or more residues chemically derivatized byreaction of a functional side group. Such derivatized molecules include,for example, those molecules in which free amino groups have beenderivatized to form amine hydrochlorides, p-toluene sulfonyl groups,carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups orformyl groups. Free carboxyl groups may be derivatized to form salts,methyl and ethyl esters or other types of esters or hydrazides. Freehydroxyl groups may be derivatized to form O-acyl or O-alkylderivatives. The imidazole nitrogen of histidine may be derivatized toform N-im-benzylhistidine. Also included as chemical derivatives arethose peptides which contain one or more naturally occurring amino acidderivatives of the twenty standard amino acids. For examples:4-hydroxyproline may be substituted for proline; 5-hydroxylysine may besubstituted for lysine; 3-methylhistidine may be substituted forhistidine; homoserine may be substituted for serine; and ornithine maybe substituted for lysine. Polypeptides of the present invention alsoinclude any polypeptide having one or more additions and/or deletions ofresidues, relative to the sequence of a polypeptide whose sequence isshown herein, so long as the required activity is maintained.

The present invention also provides compositions containing anacceptable carrier and any of an isolated, purified SMDP and/or SCPmature protein or functional polypeptide fragments thereof, alone or incombination with each other. These polypeptides or proteins can berecombinantly derived, chemically synthesized or purified from nativesources. As used herein, the term “acceptable carrier” encompasses anyof the standard pharmaceutical carriers, such as phosphate bufferedsaline solution, water and emulsions such as an oil/water or water/oilemulsion, and various types of wetting agents. The SMDP and/or SCPcompositions described herein can be used, for example, in methodsdescribed hereinafter.

Also provided are antisense-nucleic acids having a sequence capable ofbinding specifically with full-length or any portion of an mRNA thatencodes SMDP and/or SCP polypeptides so as to prevent translation of themRNA. The antisense-nucleic acid may have a sequence capable of bindingspecifically with any portion of the sequence of the cDNA encoding SMDPand/or SCP polypeptides. As used herein, the phrase “bindingspecifically” encompasses the ability of a nucleic acid sequence torecognize a complementary nucleic acid sequence and to formdouble-helical segments therewith via the formation of hydrogen bondsbetween the complementary base pairs. An example of an antisense-nucleicacid is an antisense-nucleic acid comprising chemical analogs ofnucleotides.

Compositions comprising an amount of the antisense-nucleic acid,described above, effective to reduce expression of SMDP and/or SCPpolypeptides by passing through a cell membrane and binding specificallywith mRNA encoding SMDP and/or SCP polypeptides so as to preventtranslation and an acceptable hydrophobic carrier capable of passingthrough a cell membrane are also provided herein. Suitable hydrophobiccarriers are described, for example, in U.S. Pat. Nos. 5,334,761;4,889,953; 4,897,355, and the like. The acceptable hydrophobic carriercapable of passing through cell membranes may also comprise a structurewhich binds to a receptor specific for a selected cell type and isthereby taken up by cells of the selected cell type. The structure maybe part of a protein known to bind to a cell-type specific receptor.

Antisense-nucleic acid compositions are useful to inhibit translation ofmRNA encoding invention polypeptides. Synthetic oligonucleotides, orother antisense chemical structures are designed to bind to mRNAencoding SMDP and/or SCP polypeptides and inhibit translation of mRNAand are useful as compositions to inhibit expression of SMDP and/or SCPassociated genes in a tissue sample or in a subject.

In accordance with another embodiment of the invention, kits areprovided for detecting mutations, duplications, deletions,rearrangements and aneuploidies in SMDP and/or SCP genes comprising atleast one invention probe or antisense nucleotide.

The present invention provides means to modulate levels of expression ofSMDP and/or SCP polypeptides by employing synthetic antisense-nucleicacid compositions (hereinafter SANC) which inhibit translation of mRNAencoding these polypeptides. Synthetic oligonucleotides, or otherantisense-nucleic acid chemical structures designed to recognize andselectively bind to mRNA, are constructed to be complementary tofull-length or portions of an SMDP and/or SCP coding strand, includingnucleotide sequences set forth in SEQ ID NOs:1, 3, 5, 7, 9, 11 and 13.The SANC is designed to be stable in the blood stream for administrationto a subject by injection, or in laboratory cell culture conditions. TheSANC is designed to be capable of passing through the cell membrane inorder to enter the cytoplasm of the cell by virtue of physical andchemical properties of the SANC which render it capable of passingthrough cell membranes, for example, by designing small, hydrophobicSANC chemical structures, or by virtue of specific transport systems inthe cell which recognize and transport the SANC into the cell. Inaddition, the SANC can be designed for administration only to certainselected cell populations by targeting the SANC to be recognized byspecific cellular uptake mechanisms which bind and take up the SANC onlywithin select cell populations. In a particular embodiment the SANC isan antisense oligonucleotide.

For example, the SANC may be designed to bind to a receptor found onlyin a certain cell type, as discussed supra. The SANC is also designed torecognize and selectively bind to target mRNA sequence, which maycorrespond to a sequence contained within the sequences shown in SEQ IDNOs: 1, 3, 5, 7, 9, 11 and 13. The SANC is designed to inactivate targetmRNA sequence by either binding thereto and inducing degradation of themRNA by, for example, RNase I digestion, or inhibiting translation ofmRNA target sequence by interfering with the binding oftranslation-regulating factors or ribosomes, or inclusion of otherchemical structures, such as ribozyme sequences or reactive chemicalgroups which either degrade or chemically modify the target mRNA. SANCshave been shown to be capable of such properties when directed againstmRNA targets (see Cohen et al., TIPS, 10:435 (1989) and Weintraub, Sci.American, January (1990), pp. 40; both incorporated herein byreference).

In accordance with yet another embodiment of the present invention,there is provided a method for the recombinant production of inventionSMDPs and/or SCPs by expressing the above-described nucleic acidsequences in suitable host cells. Recombinant DNA expression systemsthat are suitable to produce SMDPs and/or SCPs described herein arewell-known in the art. For example, the above-described nucleotidesequences can be incorporated into vectors for further manipulation. Asused herein, vector (or plasmid) refers to discrete elements that areused to introduce heterologous DNA into cells for either expression orreplication thereof.

Suitable expression vectors are well-known in the art, and includevectors capable of expressing DNA operatively linked to a regulatorysequence, such as a promoter region that is capable of regulatingexpression of such DNA. Thus, an expression vector refers to arecombinant DNA or RNA construct, such as a plasmid, a phage,recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the inserted DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome.

As used herein, a promoter region refers to a segment of DNA thatcontrols transcription of DNA to which it is operatively linked.Promoters, depending upon the nature of the regulation, may beconstitutive or regulated. Exemplary promoters contemplated for use inthe practice of the present invention include the SV40 early promoter,the cytomegalovirus (CMV) promoter, the mouse mammary tumor virus (MMTV)steroid-inducible promoter, Moloney murine leukemia virus (MMLV)promoter, and the like.

As used herein, expression refers to the process by which polynucleicacids are transcribed into mRNA and translated into peptides,polypeptides, or proteins. If the polynucleic acid is derived fromgenomic DNA, expression may, if an appropriate eukaryotic host cell ororganism is selected, include splicing of the mRNA.

Prokaryotic transformation vectors are well-known in the art and includepBlueskript and phage Lambda ZAP vectors (Stratagene, La Jolla, Calif.),and the like. Other suitable vectors and promoters are disclosed indetail in U.S. Pat. No. 4,798,885, issued Jan. 17, 1989, the disclosureof which is incorporated herein by reference in its entirety.

Other suitable vectors for transformation of E. coli cells include thepET expression vectors (Novagen, see U.S. Pat. No. 4,952,496), e.g.,pET11a, which contains the T7 promoter, T7 terminator, the inducible E.coli lac operator, and the lac repressor gene; and pET 12a-c, whichcontain the T7 promoter, T7 terminator, and the E. coli ompT secretionsignal. Another suitable vector is the pIN-IIIompA2 (see Duffaud et al.,Meth. in Enzymology, 153:492-507, 1987), which contains the Ipppromoter, the lacUV5 promoter operator, the ompA secretion signal, andthe lac repressor gene.

Exemplary, eukaryotic transformation vectors, include the cloned bovinepapilloma virus genome, the cloned genomes of the murine retroviruses,and eukaryotic cassettes, such as the pSV-2 gpt system [described byMulligan and Berg, Nature Vol. 277:108-114 (1979)] the Okayama-Bergcloning system [Mol. Cell Biol. Vol. 2:161-170 (1982)], and theexpression cloning vector described by Genetics Institute [Science Vol.228:810-815 (1985)], are available which provide substantial assuranceof at least some expression of the protein of interest in thetransformed eukaryotic cell line.

In accordance with another embodiment of the present invention, thereare provided “recombinant cells” containing the nucleic acid molecules(i.e., DNA or mRNA) of the present invention. Methods of transformingsuitable host cells, preferably bacterial cells, and more preferably E.coli cells, as well as methods applicable for culturing said cellscontaining a gene encoding a heterologous protein, are generally knownin the art. See, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA (1989).

Exemplary methods of transformation include, e.g., transformationemploying plasmids, viral, or bacterial phage vectors, transfection,electroporation, lipofection, and the like. The heterologous DNA canoptionally include sequences which allow for its extrachromosomalmaintenance, or said heterologous DNA can be caused to integrate intothe genome of the host (as an alternative means to ensure stablemaintenance in the host).

Host organisms contemplated for use in the practice of the presentinvention include those organisms in which recombinant production ofheterologous proteins has been carried out. Examples of such hostorganisms include bacteria (e.g., E. coli), yeast (e.g., Saccharomycescerevisiae, Candida tropicalis, Hansenula polymorpha and P. pastoris;see, e.g., U.S. Pat. Nos. 4,882,279, 4,837,148, 4,929,555 and4,855,231), mammalian cells (e.g., HEK293, CHO and Ltk⁻ cells), insectcells, and the like. Presently preferred host organisms are bacteria.The most preferred bacteria is E. coli.

In one embodiment, nucleic acids encoding the invention SMDPs and/orSCPs can be delivered into mammalian cells, either in vivo or in vitrousing suitable viral vectors well-known in the art. Suitable retroviralvectors, designed specifically for “gene therapy” methods, aredescribed, for example, in WIPO publications WO 9205266 and WO 9214829,which provide a description of methods for efficiently introducingnucleic acids into human cells. In addition, where it is desirable tolimit or reduce the in vivo expression of the invention SMDP and/or SCP,the introduction of the antisense strand of the invention nucleic acidis contemplated.

Viral based systems provide the advantage of being able to introducerelatively high levels of the heterologous nucleic acid into a varietyof cells. Suitable viral vectors for introducing invention nucleic acidencoding an SMDP and/or SCP protein into mammalian cells (e.g., vasculartissue segments) are well known in the art. These viral vectors include,for example, Herpes simplex virus vectors (e.g., Geller et al., Science,241:1667-1669 (1988)), Vaccinia virus vectors (e.g., Piccini et al.,Meth. in Enzymology, 153:545-563 (1987); Cytomegalovirus vectors(Mocarski et al., in Viral Vectors, Y. Gluzman and S. H. Hughes, Eds.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988, pp.78-84), Moloney murine leukemia virus vectors (Danos et al., PNAS, USA,85:6469 (1980)), adenovirus vectors (e.g., Logan et al., PNAS, USA,81:3655-3659 (1984); Jones et al., Cell, 17:683-689 (1979); Berkner,Biotechniques, 6:616-626 (1988); Cotten et al., PNAS, USA, 89:6094-6098(1992); Graham et al., Meth. Mol. Biol., 7:109-127 (1991)),adeno-associated virus vectors, retrovirus vectors (see, e.g., U.S. Pat.Nos. 4,405,712 and 4,650,764), and the like. Especially preferred viralvectors are the adenovirus and retroviral vectors.

For example, in one embodiment of the present invention,adenovirus-transferrin/polylysine-DNA (TfAdp1-DNA) vector complexes(Wagner et al., PNAS, USA, 89:6099-6103 (1992); Curiel et al., Hum. GeneTher., 3:147-154 (1992); Gao et al., Hum. Gene Ther., 4:14-24 (1993))are employed to transduce mammalian cells with heterologous SMDP and/orSCP nucleic acid. Any of the plasmid expression vectors described hereinmay be employed in a TfAdp1-DNA complex.

As used herein, “retroviral vector” refers to the well-known genetransfer plasmids that have an expression cassette encoding anheterologous gene residing between two retroviral LTRs. Retroviralvectors typically contain appropriate packaging signals that enable theretroviral vector, or RNA transcribed using the retroviral vector as atemplate, to be packaged into a viral virion in an appropriate packagingcell line (see, e.g., U.S. Pat. No. 4,650,764).

Suitable retroviral vectors for use herein are described, for example,in U.S. Pat. No. 5,252,479, and in WIPO publications WO 92/07573, WO90/06997, WO 89/05345, WO 92/05266 and WO 92/14829, incorporated hereinby reference, which provide a description of methods for efficientlyintroducing nucleic acids into human cells using such retroviralvectors. Other retroviral vectors include, for example, the mousemammary tumor virus vectors (e.g., Shackleford et al., PNAS, USA,85:9655-9659 (1988)), and the like.

In accordance with yet another embodiment of the present invention,there are provided anti-SMDP and/or SCP antibodies having specificreactivity with an SMDP and/or SCP polypeptides of the presentinvention. Active fragments of antibodies are encompassed within thedefinition of “antibody”. Invention antibodies can be produced bymethods known in the art using invention polypeptides, proteins orportions thereof as antigens. For example, polyclonal and monoclonalantibodies can be produced by methods well known in the art, asdescribed, for example, in Harlow and Lane, Antibodies: A LaboratoryManual (Cold Spring Harbor Laboratory (1988)), which is incorporatedherein by reference. Invention polypeptides can be used as immunogens ingenerating such antibodies. Alternatively, synthetic peptides can beprepared (using commercially available synthesizers) and used asimmunogens. Amino acid sequences can be analyzed by methods well knownin the art to determine whether they encode hydrophobic or hydrophilicdomains of the corresponding polypeptide. Altered antibodies such aschimeric, humanized, CDR-grafted or bifunctional antibodies can also beproduced by methods well known in the art. Such antibodies can also beproduced by hybridoma, chemical synthesis or recombinant methodsdescribed, for example, in Sambrook et al., supra., and Harlow and Lane,supra. Both anti-peptide and anti-fusion protein antibodies can be used.(see, for example, Bahouth et al., Trends Pharmacol. Sci. 12:338 (1991);Ausubel et al., Current Protocols in Molecular Biology (John Wiley andSons, NY (1989) which are incorporated herein by reference).

Antibody so produced can be used, inter alia, in diagnostic methods andsystems to detect the level of SMDP and/or SCP present in a mammalian,preferably human, body sample, such as tissue or vascular fluid. Suchantibodies can also be used for the immunoaffinity or affinitychromatography purification of the invention SMDP and/or SCP. Inaddition, methods are contemplated herein for detecting the presence ofan invention SMDP and/or SCP protein in a tissue or cell, comprisingcontacting the cell with an antibody that specifically binds to SMDPand/or SCP polypeptides, under conditions permitting binding of theantibody to the SMDP and/or SCP polypeptides, detecting the presence ofthe antibody bound to the SMDP and/or SCP polypeptide, and therebydetecting the presence of invention polypeptides. With respect to thedetection of such polypeptides, the antibodies can be used for in vitrodiagnostic or in vivo imaging methods.

Immunological procedures useful for in vitro detection of target SMDPand/or SCP polypeptides in a sample include immunoassays that employ adetectable antibody. Such immunoassays include, for example, ELISA,Pandex microfluorimetric assay, agglutination assays, flow cytometry,serum diagnostic assays and immunohistochemical staining procedureswhich are well known in the art. An antibody can be made detectable byvarious means well known in the art. For example, a detectable markercan be directly or indirectly attached to the antibody. Useful markersinclude, for example, radionucleotides, enzymes, fluorogens, chromogensand chemiluminescent labels.

Invention anti-SMDP and/or SCP antibodies are contemplated for useherein to modulate the activity of the SMDP and/or SCP polypeptide inliving animals, in humans, or in biological tissues or fluids isolatedtherefrom. The term “modulate” refers to a compound's ability toincrease (e.g., via an agonist) or inhibit (e.g., via an antagonist) thebiological activity of an invention SMDP and/or SCP protein, such as theparticipation in Siah-Mediated-Degradation via an SFC complex and the26S proteosome. Accordingly, compositions comprising a carrier and anamount of an antibody having specificity for SMDP and/or SCPpolypeptides effective to inhibit naturally occurring ligands or otherSMDP and/or SCP-binding proteins from binding to invention SMDP and/orSCP polypeptides are contemplated herein. For example, a monoclonalantibody directed to an epitope of an invention SMDP and/or SCPpolypeptide including an amino acid sequence set forth in SEQ ID NOs:2,4, 6, 8, 10, 12 or 14, can be useful for this purpose.

The present invention further provides transgenic non-human mammals thatare capable of expressing exogenous nucleic acids encoding SMDP and/orSCP polypeptides. As employed herein, the phrase “exogenous nucleicacid” refers to nucleic acid sequence which is not native to the host,or which is present in the host in other than its native environment(e.g., as part of a genetically engineered DNA construct). In additionto naturally occurring levels of SMDP and/or SCP, invention SMDPs and/orSCPs can either be overexpressed or underexpressed (such as in thewell-known knock-out transgenics) in transgenic mammals.

Also provided are transgenic non-human mammals capable of expressingnucleic acids encoding SMDP and/or SCP polypeptides so mutated as to beincapable of normal activity, i.e., do not express native SMDP and/orSCP. The present invention also provides transgenic non-human mammalshaving a genome comprising antisense nucleic acids complementary tonucleic acids encoding SMDP and/or SCP polypeptides, placed so as to betranscribed into antisense mRNA complementary to mRNA encoding SMDPand/or SCP polypeptides, which hybridizes to the mRNA and, thereby,reduces the translation thereof. The nucleic acid may additionallycomprise an inducible promoter and/or tissue specific regulatoryelements, so that expression can be induced, or restricted to specificcell types. Examples of nucleic acids are DNA or cDNA having a codingsequence substantially the same as the coding sequence shown in SEQ IDNOs:1, 3, 5, 7, 9, 11 and 13. An example of a non-human transgenicmammal is a transgenic mouse. Examples of tissue specificity-determiningelements are the metallothionein promoter and the L7 promoter.

Animal model systems which elucidate the physiological and behavioralroles of SMDP and/or SCP polypeptides are also provided, and areproduced by creating transgenic animals in which the expression of theSMDP and/or SCP polypeptide is altered using a variety of techniques.Examples of such techniques include the insertion of normal or mutantversions of nucleic acids encoding an SMDP and/or SCP polypeptide bymicroinjection, retroviral infection or other means well known to thoseskilled in the art, into appropriate fertilized embryos to produce atransgenic animal. (See, for example, Hogan et al., Manipulating theMouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory,(1986)).

Also contemplated herein, is the use of homologous recombination ofmutant or normal versions of SMDP and/or SCP genes with the native genelocus in transgenic animals, to alter the regulation of expression orthe structure of SMDP and/or SCP polypeptides (see, Capecchi et al.,Science 244:1288 (1989); Zimmer et al., Nature 338:150 (1989); which areincorporated herein by reference). Homologous recombination techniquesare well known in the art. Homologous recombination replaces the native(endogenous) gene with a recombinant or mutated gene to produce ananimal that cannot express native (endogenous) protein but can express,for example, a mutated protein which results in altered expression ofSMDP and/or SCP polypeptides.

In contrast to homologous recombination, microinjection adds genes tothe host genome, without removing host genes. Microinjection can producea transgenic animal that is capable of expressing both endogenous andexogenous SMDP and/or SCP. Inducible promoters can be linked to thecoding region of nucleic acids to provide a means to regulate expressionof the transgene. Tissue specific regulatory elements can be linked tothe coding region to permit tissue-specific expression of the transgene.Transgenic animal model systems are useful for in vivo screening ofcompounds for identification of specific ligands, i.e., agonists andantagonists, which activate or inhibit SMDP and/or SCP proteinresponses.

SMDP and/or SCP proteins, such as Siah-1, are contemplated herein to bea tumor suppressor proteins. Tumor suppressor proteins generally arethought to have a function in signal transduction. Mutation results inloss of function whereupon a signal pathway that the suppressor proteinregulates is left in the “on” position, which results in unregulatedcell proliferation resulting in cancerous tumor formation. Nearly alltumor suppressors regulate cell division, and proliferation, and mayhave involvement in biochemical pathways of development and the cellcycle.

The functions of the invention SMDP and/or SCP proteins support the roleof both Siah and the invention SMDPs and/or SCPs in cellular pathwaysthat affect protein degradation, such as by activating or inhibitingprotein degradation, cell division and proliferation. Accordingly,invention SMDP and/or SCP proteins provide targets for treating a broadvariety of pathologies, such as proliferative diseases, cancerpathologies, and the like.

For example, in accordance with yet another embodiment of the presentinvention, Siah-1 has been found to bind to the protein APC (Kinzler, etal., 1996, supra). The APC protein is known to bind to β-catenin andtarget it for ubiquitination and degradation (Korinek, V. et al., 1997,Science, 275:1784-1786, Rubinfeld, B. et al., 1997, Science,275:1790-1792, and Morin, P. J. et al., 1997, Science, 275:1787-1790).Defects in the regulation of the APC/β-catenin pathway for cell growthcontrol have been implicated in a variety of cancer pathologies, such asepithelial cancers, and the like. Thus, in accordance with the presentinvention Siah-1, and antagonist or agonists thereof, are contemplatedfor use in methods for treating a variety of cancers, such as epithelialcancer and the like, preferably by modulating β-catenin degradation.When used for binding to APC, fragments comprising the carboxy terminusof Siah-1, preferably comprising at least amino acids 252-298 of SEQ IDNO:2, are employed (See FIG. 3).

Invention nucleic acids, oligonucleotides (including antisense), vectorscontaining same, transformed host cells, polypeptides and combinationsthereof, as well as antibodies of the present invention, can be used toscreen compounds in vitro to determine whether a compound functions as apotential agonist or antagonist to invention polypeptides. These invitro screening assays provide information regarding the function andactivity of invention polypeptides, which can lead to the identificationand design of compounds that are capable of specific interaction withone or more types of invention proteins or fragments thereof.

Thus, in accordance with yet another embodiment of the presentinvention, there are provided methods for identifying compounds whichbind to, and preferably, modulate the activity of SMDP and/or SCPpolypeptides. The invention proteins may be employed in a competitivebinding assay. Such an assay can accommodate the rapid screening of alarge number of compounds to determine which compounds, if any, arecapable of binding to SMDPs and/or SCPs. Subsequently, more detailedassays can be carried out with those compounds found to bind, to furtherdetermine whether such compounds act as modulators, agonists orantagonists of invention SMDP and/or SCP proteins. Compounds that bindto and/or modulate invention SMDPs and/or SCPs can be used to treat avariety of pathologies mediated by invention SMDPs and/or SCPs, asdescribed herein.

In accordance with another embodiment of the present invention,transformed host cells that recombinantly express invention polypeptidescan be contacted with a test compound, and the modulating effect(s)thereof can then be evaluated by comparing the SMDP-mediated response(e.g., the degradation of a known Siah-mediated target, such as DCC orβ-catenin) in the presence and absence of test compound, or by comparingthe response of test cells or control cells (i.e., cells that do notexpress SMDP and/or SCP polypeptides), to the presence of the compound.

As used herein, a compound or a signal that “modulates the activity” ofinvention polypeptides refers to a compound or a signal that alters theactivity of SMDP and/or SCP polypeptides so that the activity of theinvention polypeptide is different in the presence of the compound orsignal than in the absence of the compound or signal. In particular,such compounds or signals include agonists and antagonists. An agonistencompasses a compound or a signal that activates SMDP and/or SCPprotein expression. Alternatively, an antagonist includes a compound orsignal that interferes with SMDP and/or SCP expression. Typically, theeffect of an antagonist is observed as a blocking of agonist-inducedprotein activation. Antagonists include competitive and non-competitiveantagonists. A competitive antagonist (or competitive blocker) interactswith or near the site specific for agonist binding. A non-competitiveantagonist or blocker inactivates the function of the polypeptide byinteracting with a site other than the agonist interaction site.

As understood by those of skill in the art, assay methods foridentifying compounds that modulate SMDP and/or SCP activity generallyrequire comparison to a control. For example, one type of a “control” isa cell or culture that is treated substantially the same as the testcell or test culture exposed to the compound, with the distinction thatthe “control” cell or culture is not exposed to the compound. Anothertype of “control” cell or culture may be a cell or culture that isidentical to the transfected cells, with the exception that the“control” cell or culture do not express native proteins. Accordingly,the response of the transfected cell to compound is compared to theresponse (or lack thereof) of the “control” cell or culture to the samecompound under the same reaction conditions.

Accordingly, in accordance with another embodiment of the presentinvention, there is provided a bioassay for evaluating whether testcompounds are capable of acting as agonists or antagonists for SMDPand/or SCP proteins, wherein said bioassay comprises: (a) culturingcells containing: DNA which expresses an SMDP and/or SCP or functionalfragments thereof, wherein said culturing is carried out in the presenceof at least one compound whose ability to modulate an activity of anSMDP and/or SCP is sought to be determined, wherein said activity isselected from a protein:protein binding activity or a proteindegradation activity and thereafter (b) monitoring said cells for eitheran increase or decrease in the level of protein:protein binding orprotein degradation.

Methods well-known in the art for measuring protein:protein binding orprotein degradation can be employed in bioassays described herein toidentify agonists and antagonists of SMDP and/or SCP proteins. Forexample, the Siah-1 over-expression assay described in Example 14 can beused to evaluate the cell degradation activity of recombinant SMDPand/or SCP proteins or mutants and/or analogs thereof, expressed inmammalian host cells.

As used herein, “ability to modulate protein degradation activity of anSMDP and/or SCP” protein refers to a compound that has the ability toeither induce (agonist) or inhibit (antagonist) the protein degradationactivity of SMDP and/or SCP proteins within a cell. Host cellscontemplated for use in the bioassay(s) of the present invention includehuman and other mammalian cells (readily available from American TypeCulture Collection), as well as genetically engineered yeast or bacteriathat express human SMDPs and/or SCPs, and the like.

In yet another embodiment of the present invention, there are providedmethods for modulating the protein degradation activity mediated by SMDPand/or SCP protein(s), said method comprising:

contacting an SMDP and/or SCP protein with an effective, modulatingamount of an agonist or antagonist identified by the above-describedbioassays.

Also provided herein are methods of treating pathologies, said methodcomprising administering an effective amount of an invention therapeuticcomposition. Such compositions are typically administered in aphysiologically compatible composition.

Exemplary diseases related to abnormal cell proliferation contemplatedherein for treatment according to the present invention include cancerpathologies, keratin hyperplasia, neoplasia, keloid, benign prothetichypertrophy, inflammatory hyperplasia, and the like. Exemplary cancerpathologies contemplated herein for treatment include, gliomas,carcinomas, sarcomas, melanomas, hamartomas, leukemias, lymphomas, andthe like.

Also contemplated herein, are therapeutic methods using inventionpharmaceutical compositions for the treatment of pathological disordersin which there is too little cell division, such as, for example, bonemarrow aplasias, immunodeficiencies due to a decreased number oflymphocytes, and the like. Methods of treating a variety of inflammatorydiseases with invention therapeutic compositions are also contemplatedherein, such as treatment of sepsis, fibrosis (e.g., scarring),arthritis, graft versus host disease, and the like.

Accordingly, the present invention contemplates therapeutic compositionsuseful for practicing the therapeutic methods described herein.Therapeutic compositions of the present invention, such aspharmaceutical compositions, contain a physiologically compatiblecarrier together with an invention SMDP and/or SCP (or functionalfragment thereof), a SMDP and/or SCP modulating agent, such as acompound (agonist or antagonist) identified by the methods describedherein, or an anti-SMDP and/or SCP antibody, as described herein,dissolved or dispersed therein as an active ingredient. In a preferredembodiment, the therapeutic composition is not immunogenic whenadministered to a mammal or human patient for therapeutic purposes.

As used herein, the terms “pharmaceutically acceptable”,“physiologically compatible” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset, and thelike.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well known in the art.Typically such compositions are prepared as injectables either as liquidsolutions or suspensions; however, solid forms suitable for solution, orsuspension, in liquid prior to use can also be prepared. The preparationcan also be emulsified.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient inamounts suitable for use in the therapeutic methods described herein.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like, as well as combinations of any two or morethereof. In addition, if desired, the composition can contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents, and the like, which enhance the effectiveness ofthe active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable nontoxic salts include the acid additionsalts (formed with the free amino groups of the polypeptide) that areformed with inorganic acids such as, for example, hydrochloric acid,hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, phosphoric acid, acetic acid, propionic acid, glycolicacid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinicacid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid,naphthalene sulfonic acid, sulfanilic acid, and the like.

Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and the like; and organic bases such asmono-, di-, and tri-alkyl and -aryl amines (e.g., triethylamine,diisopropyl amine, methyl amine, dimethyl amine, and the like) andoptionally substituted ethanolamines (e.g., ethanolamine,diethanolamine, and the like).

Physiologically tolerable carriers are well known in the art. Exemplaryliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH, physiological saline orboth, such as phosphate-buffered saline. Still further, aqueous carrierscan contain more than one buffer salt, as well as salts such as sodiumand potassium chlorides, dextrose, polyethylene glycol and othersolutes.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary additional liquid phases includeglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

As described herein, an “effective amount” is a predetermined amountcalculated to achieve the desired therapeutic effect, e.g., to modulatethe protein degradation activity of an invention SMDP and/or SCPprotein. The required dosage will vary with the particular treatment andwith the duration of desired treatment; however, it is anticipated thatdosages between about 10 micrograms and about 1 milligram per kilogramof body weight per day will be used for therapeutic treatment. It may beparticularly advantageous to administer such compounds in depot orlong-lasting form as discussed hereinafter. A therapeutically effectiveamount is typically an amount of an SMDP and/or SCP-modulating agent orcompound identified herein that, when administered in a physiologicallyacceptable composition, is sufficient to achieve a plasma concentrationof from about 0.1 μg/ml to about 100 μg/ml, preferably from about 1.0μg/ml to about 50 μg/ml, more preferably at least about 2 μg/ml andusually 5 to 10 μg/ml. Therapeutic invention anti-SMDP and/or SCPantibodies can be administered in proportionately appropriate amounts inaccordance with known practices in this art.

Also provided are systems using invention SMDPs and/or SCPs, orfunctional fragments thereof, for targeting any desired protein forubiquitination and degradation, thus enabling novel gene discoverythrough functional genomics strategies or providing the basis forablating target proteins involved in diseases for therapeutic purposes.

In accordance with another embodiment of the invention, there areprovided methods for “inducing the degradation of the function” of adesired protein from a particular cell or cell-system. As used hereinthe phrase “inducing the degradation of the function” refers todeleting, altering, modifying and/or degrading a target protein so thatit no longer has the ability to perform its native physiologicalfunction. This method is useful to determine the physiological/cellularfunction of the degraded protein. Thus, this invention method is usefulin alleviating one of the rate limiting steps in functional genomics.

The invention methods take advantage of the invention SMDP and/or SCPproteins or other protein-degradation binding domains provided herein tocreate a system that targets specific proteins for degradation byrecruiting them to a SCF complex for ubiquitination and subsequentdegradation by an appropriate proteosome, such as 26S proteosome. First,a protein or peptide fragment is selected that binds the proteintargeted for degradation in a cell, which is referred to herein as the“target-protein binding domain.” Such protein or peptide fragment couldbe, for example: a domain of any known protein that interacts with thetarget protein; the Fab region of an anti-target-protein antibody (e.g.,sFv, and the like); or a peptide aptamer obtained by screening (using,for example, yeast two-hybridization, phage display, or other screeningmethods) a random library of peptide aptamers. The target-proteinbinding domain is then fused (by engineering cDNAs in expressionplasmids to form a chimera) with an appropriate protein-degradationbinding domain selected from an invention SMDP and/or SCP, such as Siah,SIP, SAF-1, SAF-2 or SAD, and the like; or other known proteins involvedin protein degradation, such as F-box containing proteins, (e.g., Skp1,and the like), SOCS-box containing proteins (see, e.g., Kamura et al.,1998, Genes Dev, 12:3872; and Starr and Hilton, 1999, Bioessays, 21:47),HECT family proteins (see, e.g., Huibregtse et al. 1995 Proc. Natl.Acad. Sci. USA 92:2563-2567), or any other subunit of an E3 ubiquitinligase complex, and the like (see, e.g., Tyers and Williams, 1999,Science, 284:601-604; incorporated herein by reference in its entirety).

Similarly, with respect to ubiquitin-independent protein degradationinvolving ornithine decarboxylase (ODC), a “target-protein bindingdomain” is a protein or peptide fragment that binds the protein targetedfor degradation in a cell. When fused with an appropriateprotein-degradation binding domain of ODC, the target-protein bindingdomain binds the target protein, and degradation of the target proteinis induced.

As used herein, the phrase “protein-degradation binding domain” refersto a protein region that functions to recruit the target protein into amember of the superfamily of E3 ubiquitin ligase complexes, such as theSCF complex, or to Siah-family proteins which may target some proteinsfor degradation independently of SCF complexes, where theprotein-degradation binding domain and/or the target protein becomeubiquitinated and then degraded, such as by the 26S proteosome,lysosomes and/or vacuoles (see, e.g., Tyers and Williams, 1999, Science,284:601-604; and Ciechanover, 1998, supra). Exemplaryprotein-degradation domains can be obtained from a protein member of theubiquitin-mediated protein-degradation family, for example, SIP, Siah,E7, Fwb7, UB1, Ub4, S5a, antizyme, and others, as disclosed herein andwell known to those skilled in the art. As used herein, the phrase “aprotein member of the ubiquitin-mediated protein-degradation family”refers to one of the numerous proteins that are known to interact, viaprotein:protein binding, in the ubiquitin system of intracellularprotein degradation (see, e.g., Ciechanover et al., 1998, supra; andTyers and Williams, supra, and the like).

Similarly, with respect to ubiquitin-independent protein degradationinvolving ODC, a “protein-degradation binding domain” is a proteinregion of ODC that functions to recruit the target protein fordegradation by the 26S proteasome. As discussed below, a full-length ODCsequence or a functional fragment of ODC, e.g., an ODC C-terminus,functions as a “protein-degradation binding domain.”

In yet another embodiment contemplated by the present invention, methodsare provided of identifying a nucleic acid molecules encoding a chimericprotein that modulates a cellular phenotype, said method comprising: (a)expressing, in a cell, a chimeric nucleic acid comprising a member of anucleic acid library fused to nucleic acid encoding a proteindegradation binding domain of a protein member of the ubiquitin-mediatedprotein degradation family; and (b) screening said cells for amodulation of said phenotype.

For example, unbiased nucleic acid libraries can be constructed wherein,each member of the nucleic acid library is expressed as an encodedprotein fused to a particular protein-degradation binding domain, suchas invention SMDPs and/or SCPs, e.g., Siah-1, SIP (see, e.g., Example15), SAF-1, SAF-2, or SAD; or other known proteins involved in proteindegradation, such as Skp1, F-box containing proteins, HECT familyproteins, or any other subunit of an E3 ubiquitin ligase complex, andthe like. As used herein a “nucleic acid library” comprises cDNAlibraries, YAC libraries, BAC libraries, cosmid libraries, or any othersource of nucleic acid encoding polypeptides. The chimeric nucleic acidencoding these fusion proteins is then introduced into cells possessinga particular phenotype to be assayed.

The cells are then subjected to a “screening” step which comprisesselecting one or more cells in which the desired phenotype has beenmodulated (e.g., suppressed or enhanced). The phenotypes to be screenedmay be any chemical or physical representation of a cellular process,including but not limited to: cell proliferation in either an attachedor detached (i.e., anchorage-independent) state, cell survival, celldeath, cell secretion, cell migration, abnormal cell morphology,chemical reactivity (e.g., heavy metals, antibiotics, etc.), physicalreactivity (e.g., heat, light, radiation, etc.), and the like.

Next, cDNAs are identified and isolated whose expression productsfunction to modulate the desired phenotype within cells. The cDNAsidentified by the invention method encode an invention chimeric proteinthat interacts, preferably by direct binding, with another protein inthe cells that is targeted for degradation, thereby eliminating itsphysiological function. Accordingly, any target-protein that has one ormore protein-binding or protein-interacting partners, or whichhomodimerizes/homo-oligomerizes is contemplated for degradation in theinvention methods. The cDNA identified by the above-described method canbe used to perform a two-hybrid screen, as described herein, to identifythe protein-binding region of the partner to the target protein, ordirectly sequenced to determine the identity of the target protein ifhomo-dimerization or homo-oligomerization situation occurs.

Accordingly, also provided in accordance with the present invention arechimeric proteins, and encoding nucleic acids, comprising atarget-protein binding domain operatively linked to aprotein-degradation binding domain of a protein member of theubiquitin-mediated protein-degradation family, or to aprotein-degradation binding domain of ODC.

Exemplary proteins whose function can be targeted for degradationaccording to the invention methods, include any protein encoded by aknown gene or cDNA whose function is desired. Exemplary targets include,for example, apoptosis-related proteins, cell-cycle regulatory proteins,heat shock proteins, transcription factors, or any other target proteinwhich, when degraded, will modulate the phenotype of a cell.

Also provided are methods for treating a disease by degrading thefunction of a target protein, comprising introducing, into a cell, achimeric protein comprising a target-protein binding domain operativelylinked to a protein-degradation binding domain of a protein member ofthe ubiquitin-mediated protein-degradation family, or to aprotein-degradation binding domain of ODC. For example, for a variety ofproteins which, when expressed in overabundant or mutated form (e.g., anoncoprotein such as ras, or a genetic mutation, such as in the CF gene(cystic fibrosis gene) result in a known pathology, the chimeric proteinof the invention may be used to therapeutically treat the disease, byway of reducing or completely eliminating, via protein degradation, thepathology causing protein. This treatment comprises fusion of a proteindomain which binds the target pathology causing protein (i.e., theprotein which causes the illness) with a particular protein-degradationbinding domain as described herein. This chimeric protein may then bedelivered to the location of the protein which causes the illness byintravenous therapy or gene therapy employing the methods describedherein, or any other method well-known to one skilled in the art fordelivering a protein to its binding target. As used herein, “treatmentof a disease” refers to a reduction in the effects of the disease,including reducing the symptoms of the disease. In accordance withanother embodiment of the present invention, there are provided methodsfor diagnosing cancer, said method comprising detecting, in saidsubject, a defective sequence or mutant of SEQ ID NOs: 1, 3, 5, 7, 9, 11and 13.

In accordance with another embodiment of the present invention, thereare provided diagnostic systems, preferably in kit form, comprising atleast one invention nucleic acid in a suitable packaging material. Thediagnostic nucleic acids are derived from the

SMDP and/or SCP-encoding nucleic acids described herein. In oneembodiment, for example, the diagnostic nucleic acids are derived fromany of SEQ ID NOs: 1, 3, 5, 7, 9, 11 and 13. Invention diagnosticsystems are useful for assaying for the presence or absence of nucleicacid encoding SMDP and/or SCP in either genomic DNA or in transcribednucleic acid (such as mRNA or cDNA) encoding SMDP and/or SCP.

A suitable diagnostic system includes at least one invention nucleicacid, preferably two or more invention nucleic acids, as a separatelypackaged chemical reagent(s) in an amount sufficient for at least oneassay. Instructions for use of the packaged reagent are also typicallyincluded. Those of skill in the art can readily incorporate inventionnucleic probes and/or primers into kit form in combination withappropriate buffers and solutions for the practice of the inventionmethods as described herein.

As employed herein, the phrase “packaging material” refers to one ormore physical structures used to house the contents of the kit, such asinvention nucleic acid probes or primers, and the like. The packagingmaterial is constructed by well known methods, preferably to provide asterile, contaminant-free environment. The packaging material has alabel which indicates that the invention nucleic acids can be used fordetecting a particular sequence encoding SMDP and/or SCP including thenucleotide sequences set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11 and 13or mutations or deletions therein, thereby diagnosing the presence of,or a predisposition for, cancer. In addition, the packaging materialcontains instructions indicating how the materials within the kit areemployed both to detect a particular sequence and diagnose the presenceof, or a predisposition for, cancer.

The packaging materials employed herein in relation to diagnosticsystems are those customarily utilized in nucleic acid-based diagnosticsystems. As used herein, the term “package” refers to a solid matrix ormaterial such as glass, plastic, paper, foil, and the like, capable ofholding within fixed limits an isolated nucleic acid, oligonucleotide,or primer of the present invention. Thus, for example, a package can bea glass vial used to contain milligram quantities of a contemplatednucleic acid, oligonucleotide or primer, or it can be a microtiter platewell to which microgram quantities of a contemplated nucleic acid probehave been operatively affixed.

“Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter, such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions, and the like.

Ornithine Decarboxylase-Mediated Protein Degradation by aUbiquitin-Independent Mechanism

Also provided are compositions and methods for targeting the destructionof selected polypeptides in eukaryotic cells based on theubiquitin-independent mechanism by which ornithine decarboxylase (ODC)is degraded by the 26S proteasome. Expressing whole polypeptides,polypeptide domains, or peptide ligands fused to the carboxy (C)terminus of ODC promotes proteasome-dependent degradation of thesechimeric fusion polypeptides and their interacting cellular targetpolypeptides. In some cases, the degradation of the interacting(targeted) polypeptide depends on co-expression of antizyme (AZ),providing an inducible switch for triggering the degradation process.The ODC/AZ system is useful for ablating expression of specificendogenous polypeptides and creates the expected lesions in cellularpathways that require these polypeptides. Thus this approach provides anadditional tool for revealing the cellular phenotypes of gene productsand for therapeutics based on destruction of targeted polypeptides.

AZ binding to ODC induces exposure of the carboxy (C) terminus of ODCand accelerates its degradation by 50- to 100-fold. Normally, thispolypeptide degradation pathway is induced in response to polyamines(e.g., spermine, spermidine, putresine, and known polyamine analogues),which trigger AZ production, thus providing a negative feedback loop formaintaining appropriate intracellular levels of these molecules(reviewed in Coffino, Nat. Rev. Mol. Cell Biol. 2:188-194, 2001). Anumber of polyamine analogs are known to those of ordinary skill in theart and may be used in the practice of the present invention.

As used herein, the terms “C terminus of ODC,” “ODC C terminus,”“C-terminal region of ODC” or “ODC C-terminal region” and similar termsare used interchangeably to refer to any ODC polypeptide or functionalfragment thereof (or a polynucleotide that encodes such a polypeptide)that binds the 26S proteasome and has ODC activity. Thus, an ODC Cterminus functions as a “protein-degradation binding domain” as definedherein, although the resulting protein degradation is notubiquitin-mediated. The term “ODC activity” refers to the activity of aC terminus ODC in directing the degradation of a target polypeptide bythe 26S proteasome when the C terminus of ODC is fused to atarget-protein binding domain, such as a ligand of the targetpolypeptide, directly or indirectly through a linker separating theligand and the C terminus of ODC. An ODC C terminus optionally alsoincludes a region of the ODC polypeptide required for homodimerization.A C terminus of ODC includes a full-length ODC polypeptide or a fragmentof a full-length ODC polypeptide. Wild-type, or native, ODCpolypeptide-encoding polynucleotides and fragments thereof may beobtained from any eukaryotic source, including but not limited to humanor other mammalian cells. Alternatively, modified (including one or morenucleotide sequence changes from a reference wild-type sequence, e.g.,by known mutagenesis techniques) and synthetic (made with a desiredsequence that is different from a wild-type sequence, e.g., using a DNAsynthesizer) ODC C terminus polypeptides may be used in the practice ofthe invention.

As used herein, the term “antizyme” (AZ) includes a full-length AZpolypeptide or any fragment of AZ that has AZ activity (or apolynucleotide that encodes such a polypeptide). The term “AZ activity”refers to the activity of AZ in binding ODC and inducing exposure of theC terminus of ODC, thereby catalyzing, i.e., increasing the rate of,degradation of ODC by the 26S proteasome. A full-length antizymepolypeptide may be employed. Alternatively, a fragment of antizyme thatcatalyzes ODC degradation may be employed. Wild-type AZ polypeptide andfragments thereof may be obtained from any eukaryotic source, includingbut not limited to human AZ. Alternatively, modified (non-native) AZpolypeptides may be used in the practice of the invention.

As used herein, the term “linker” includes any polypeptide or peptidesequence that is included between a ligand sequence and an ODC Cterminus sequence in a fusion polypeptide according to the presentinvention (or a polynucleotide that encodes such a polypeptide). Anylinker polypeptide that permits degradation, or preferably increasesdegradation, of a target protein, may be used in the practice of theinvention. Wild-type linker polypeptides or peptides may be obtainedfrom any source. Alternatively, modified (non-native) or syntheticlinker polypeptides may be used in the practice of the invention. Avariety of flexible linker (FL) sequences that increase the efficiencyof degradation of target polypeptides are disclosed in the Examplebelow.

Therefore, according to one embodiment of the invention, a polypeptideis targeted for destruction in a eukaryotic cell by expression in thecell of a fusion polypeptide that comprises a target-protein bindingdomain and a protein-degradation binding domain of ODC, i.e., a Cterminus of ODC.

One embodiment of the invention is directed to determining the functionof a polypeptide of interest that is encoded by a recombinant expressionvector according to the present invention. According to one embodimentof the invention, the fusion polypeptide includes a linker between thetarget-protein binding domain and the protein-degradation bindingdomain, i.e., the ODC C terminus. According to another embodiment of theinvention, such a fusion polypeptide comprises, in sequence from itsamino (N) terminus to its carboxy (C) terminus, the target-proteinbinding domain, the linker portion, and the protein-degradation bindingdomain. According to a further embodiment, the fusion polypeptideincludes an epitope tag located anywhere in the fusion polypeptide thatdoes not interfere with function of the fusion polypeptide, to allowverification of protein production by immunoblotting and/or confirmationof binding to cellular target proteins by co-immunoprecipitation assays.When introduced into a host cell, the target-protein binding domaininteracts with (e.g., binds to) the targeted polypeptide of interest,and ODC binds to the 26S proteasome, leading to degradation of thefusion polypeptide and the target polypeptide by the 26S proteasome. Theresulting degradation of the targeted polypeptide of interest isdetected by detection of a reduction in intracellular levels of thepolypeptide of interest or changes in one or more phenotypes associatedwith such a reduction in levels of the polypeptide.

Exemplary polypeptides that can be targeted for degradation according tothe methods of the present invention include any polypeptide, including,but not limited to, polypeptides involved in apoptosis, regulation ofthe cell cycle, heat shock, transcription factors, etc.

According to one embodiment of the invention, an expression vector thatencodes and expresses a chimeric fusion polypeptide of the presentinvention is introduced into eukaryotic (e.g., mammalian) cells invitro. According to another embodiment of the invention, such anexpression vector is introduced into a eukaryotic cell, such as amammalian embryonic stem (ES) cell or a fertilized or unfertilized eggcell, and the cell with the introduced expression vector develops intoan organism, each cell of which includes the introduced expressionvector. According to another embodiment of the invention, such anexpression vector is introduced into a cell of a eukaryotic organism invivo. According to another embodiment of the invention, such anexpression vector is introduced into cells of an organism (e.g., bonemarrow cells or isolated stem cells) ex vivo, then the cells areintroduced into the organism. Accordingly, the present inventionprovides compositions that comprise such polynucleotides, includingpharmaceutical compositions comprising such polynucleotides and apharmaceutically acceptable carrier.

In another embodiment of the invention, isolated chimeric fusionpolypeptides according to the invention are administered to cells inwhich a polypeptide of interest is expressed. Upon uptake by the cells,the fusion polypeptide binds to the polypeptide of interest, targetingits degradation by the 26S proteasome and reducing levels of thepolypeptide of interest in the cells. Accordingly, the present inventionprovides compositions that comprise such fusion polypeptides, such aspharmaceutical compositions comprising such fusion polypeptides and apharmaceutically acceptable carrier.

In another embodiment of the invention, methods are provided ofidentifying a polynucleotide that modulates a phenotype of a cell. Asused herein, the term “modulates” is used to refer to any increase ordecrease in the magnitude of a phenotype, or the appearance ordisappearance of a phenotype, or any other detectable change in aphenotype of a cell (or a tissue, organ, or organism comprising such acell). Such methods comprise: (a) introducing into the cell an isolatedpolynucleotide comprising a expression control sequence and aprotein-coding sequence, operably linked to the expression controlsequence, that encodes a polypeptide that comprises a ligand sequencethat binds to a cellular polypeptide that is not degraded by a 26Sproteasome in a cell comprising the cellular polypeptide and a targetingsequence, wherein expression of the polynucleotide in the cell causesthe cellular polypeptide to be degraded by the 26S proteasome; and (b)observing a modulation of phenotype of the cell. In such methods ofidentifying, or screening for, polynucleotides that modulate a phenotypeof a cell, for example, unbiased polynucleotide libraries can bescreened, including but not limited to cDNA, cosmid, yeast artificialchromosome (YAC), or bacterial artificial chromosome (BAC) libraries, orany other source of polynucleotides. The phenotype to be screened may beany chemical or physical representation of a cellular process, includingbut not limited to: proliferation in an attached or detached (i.e.,anchorage dependent or independent) state, survival, death, secretion,migration, morphology, reactivity to chemical substances (e.g., to heavymetals, antibiotics, etc.) or physical stimuli (e.g., heat, light,radiation, etc.), and the like. A polynucleotide that causes a modulatein phenotype of a cell in such an assay can be used to perform atwo-hybrid screen to determine the protein-binding region of the partnerto the targeted protein or directly sequenced to determine the identityof the target protein if homo-dimerization or homo-oligomerizationoccurs.

Organisms useful for functional studies with recombinant polynucleotidesaccording to the present invention are those in which the polynucleotideis able to inhibit expression of a target polypeptide in cells thatexpress the target polypeptide. Organisms include an embryo, a juvenilestage, or an adult animal, including, but not limited to, a fish, afrog, a mouse, a rat, guinea pig, a sheep, a monkey, or a human. As usedherein, the term “cell” refers to an isolated cell or cells in vitro,tissues, organs, or whole organisms.

Similarly, polynucleotides according to the present invention also areuseful for generating model organisms for the study of diseases. Diseaseconditions associated with loss or reduction of function of a particularpolypeptide in a higher organism such as a human can be generated in amodel organism if the model organism has a homologous or orthologouspolypeptide. For example, a polynucleotide according to the presentinvention that targets the homologue or orthologue in the model organismis introduced into the model organism (such as a mouse, for example),thereby creating a “knock out” of the homologue or orthologue. Reductionin expression of the homologue or orthologue present in the modelorganism results in a morphant organism that exhibits the diseasecondition or other detectable phenotype resulting from the loss orreduction in function of the homologous or orthologous polypeptide. Suchmorphants can be used to screen for compounds that are useful fortreating the disease condition or alleviating the severity of thedisease condition. To screen for compounds that are useful for treatinga disease condition or alleviating the severity of the diseasecondition, morphants exhibiting the disease condition can be contactedwith candidate compounds and then assessed to determine whether thedisease condition is lessened.

Polynucleotides according to the present invention also can be used toidentify the function of polypeptides not known to be associated with adisease condition. To identify polypeptides not known to be associatedwith a disease condition, a polynucleotide according to the presentinvention is introduced into a wild-type organisms and morphantsexhibiting any particular disease condition or other detectablephenotype are chosen for further analysis. A collection ofpolynucleotides according to the present invention can be used togenerate a collection of morphants that can, in turn, be used toidentify drug targets as well as to develop novel treatments for adisease condition. A potential drug target, for example, is identifiedas the polypeptide whose reduction in expression led to the diseasecondition.

To identify polypeptides whose excessive activity leads to a diseasecondition, polynucleotides according to the present invention areintroduced into organisms exhibiting the disease condition. Thoseorganisms whose disease state is lessened by the polynucleotides arefurther examined to identify polynucleotides whose expressions have beenreduced. These polynucleotides are identified as useful targets for thedevelopment of novel treatments for the particular disease.

Expression vectors according to the present invention also can be usedtherapeutically as treatments for disease conditions. In order to treata disease condition associated with expression (or overexpression) of aparticular polypeptide, an expression vector that expresses a chimericfusion polypeptide according to the present invention is introduced intoan organism in need of treatment by any known means, as discussed below.According to one embodiment of the invention, such an expression vectoris introduced into a cell of a eukaryotic organism in vivo. According toanother embodiment of the invention, such an expression vector isintroduced into cells of an organism (e.g., bone marrow cells orisolated stem cells) ex vivo, then the cells are introduced into theorganism. Upon introduction into cells and expression within the cells,a cellular polypeptide is targeted for destruction, providing atherapeutic benefit. For example, such targeted polypeptides may includepolypeptides that, when inappropriately expressed (e.g., over- orunderexpressed or expressed or expressed at an abnormal time orlocation) or expressed in a mutated form, cause a disease symptom in apatient, including oncoproteins (e.g., ras), cellular receptors, iontransporters or channels (e.g., the cystic fibrosis gene), etc.

All U.S. patents and all publications mentioned herein are incorporatedin their entirety by reference thereto. The invention will now bedescribed in greater detail by reference to the following non-limitingexamples.

EXAMPLES

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., MolecularCloning: A Laboratory Manual (2 ed.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1989); Davis et al., Basic Methodsin Molecular Biology, Elsevier Science Publishing, Inc., New York, USA(1986); or Methods in Enzymology: Guide to Molecular Cloning TechniquesVol. 152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., SanDiego, USA (1987)).

Library screening by the yeast two-hybrid method was performed herein asdescribed (Durfee et al., 1993; Sato et al., 1995; Matsuzawa et al.1998) using the pGilda plasmid encoding the desired amino acid region asbait, an appropriate cDNA library, and the EGY48 strain S. cerevisiae(MATa, trp1, ura3, his, leu2::plexApo6-leu2). Cells were grown in eitherYPD medium with 1% yeast extract, 2% polypeptone, and 2% glucose, or inBurkholder's minimal medium (BMM) fortified with appropriate amino-acidsas described previously (Sato et al., 1994). Transformations wereperformed by a LiCl method using 0.25 mg of pJG4-5-cDNA library DNA, and5 mg of denatured salmon sperm carrier DNA. Clones that formed on Leudeficient BMM plates containing 2% galactose/1% raffinose weretransferred to BMM plates containing leucine and 2% glucose, and filterassays were performed for β-galactosidase measurements as previouslydescribed.

Example 1 Yeast Two-Hybrid Screen of BAG-1 Binding Proteins to ObtaincDNA Encoding Siah-1α

The mouse BAG-1 amino acid sequence was cloned into the pGilda plasmidand used as bait to screen a human Jurkat T-cell cDNA library. From aninitial screen of ˜1.6×10⁷ transformants, 298 clones were identifiedthat trans-activated the LEU2 reporter gene based on ability to grow onleucine-deficient media. Of those, 30 colonies were also positive forβ-galactosidase. These 30 candidate transformants were then cured of theLexA/BAG-1 bait plasmid by growth in media containing histidine and thenmated with each of 5 different indicator strains of cells containing oneof following LexA bait proteins: BAG-1 (1-219), Bax (1-171), v-Ras, Fas(191-335), or Lamin-C. The mating strain was RFY206 (MATa, his3D200,leu2-3, lys2D201, ura3-52, trpID::hisG), which had been transformed withpGilda-BAG-1 or various control proteins and selected onhistidine-deficient media. This resulted in 23 clones which displayedspecific two-hybrid binding interactions with BAG-1. DNA sequencinganalysis revealed 4 cDNAs encoding portions of Siah-1.

Example 2 Isolation of Full-Length Human Siah-1α cDNAs

To obtain the complete sequence of human Siah-1, cDNA fragmentscontaining the 5′ end of human Siah 1 were PCR-amplified from Jurkatrandomly primer cDNAs by using a forward primer 5′GGGAATTCGGACTTATGGCATGTAAACA-3′ (SEQ ID NO:42) containing an EcoRI siteand a reverse primer 5′ TAGCCAAGTTGCGAATGGA-3′ (SEQ ID NO:43), based onsequences of EST database clones (NCBI ID: AA054272, AA258606, AA923663,AA418482, and AI167464). The PCR products were digested with EcoRI andBamHI, then directly subcloned into the EcoRI and SalI sites of pcIplasmid into which the cDNA derived from pJG4-5-Siah (22-298) hadpreviously been cloned, as a BamHI-XhoI fragment. The complete humanSiah-1α cDNA and amino acid sequence is set forth in SEQ ID Nos: 1 and2, respectively. The human Siah-1α sequence contains 16 N-terminal aminoacids that are not present in the human Siah-1β protein.

Example 3 Yeast Two-Hybrid Screen of Siah-1 Binding Proteins to ObtaincDNA Encoding SIP-L and SIP-S

Human Siah-1α cDNA encoding amino acids 22-298 of SEQ ID NO:1(corresponding to amino acids 6-282 set forth in Nemani et al., supra)was cloned into the pGilda plasmid and used as a bait to screen a humanembryonic brain cDNA library (Invitrogen) in EGY48 strain S. cerevisiae.From an initial screen of ˜2.0×10⁷ transformants, 322 clones wereidentified that trans-activated the LEU2 reporter gene based on abilityto grow on leucine-deficient media. Of those, 32 colonies were alsopositive for β-galactosidase. These 32 candidate transformants were thencured of the LexA/Siah-1 bait plasmid by growth in media containinghistidine and then mated with each of 5 different indicator strains ofcells containing one of following LexA bait proteins: Siah-1 (22-298),Bax (1-171), v-Ras, Fas (191-335), or BAG-1. The mating strain wasRFY206 which had been transformed with pGilda-Siah-1 or various controlproteins and selected on histidine-deficient media. This resulted in 11clones which displayed specific two-hybrid interactions with Siah-1. DNAsequencing analysis revealed 5 cDNAs encoding portions of SIP-L, 1 cDNAencoding portions of SIP-S, 3 cDNAs encoding portions of ofAPC(2681-2843), and 2 cDNAs encoding portions of Siah-1. The SIP-L andSIP-S clones were sequenced and the resulting nucleotide sequences areset forth in SEQ ID Nos:3 and 5, respectively.

Example 4 Yeast Two-Hybrid Screen of Skp1 Binding Proteins to ObtaincDNA Encoding SAF-1 and SAD

Human Skp1 cDNA encoding amino acids 91-163 of (Zhang et al., 1995,Cell, 82:915-925) was cloned into the pGilda plasmid as a bait to screena human embryonic brain cDNA library (Invitrogen) in EGY48 strain S.cerevisiae. From an initial screen of ˜1.2×10⁸ transformants, 130 cloneswere identified that trans-activated the LEU2 reporter gene based onability to grow on leucine-deficient media. Of those, 36 colonies werealso positive for β-galactosidase. These 36 candidate transformants werethen cured of the LexA/BAG-1 bait plasmid by growth in media containinghistidine and then mated with each of 5 different indicator strains ofcells containing one of following LexA bait proteins: Skp1 (91-163),SIP-L, Bax (1-171), v-Ras, Fas (191-335), or Siah-1. The mating strainwas RFY206 which had been transformed with pGilda-Skp1 or variouscontrol proteins and selected on histidine-deficient media. Thisresulted in 3 clones which displayed specific two-hybrid interactionswith Skp1 and 18 clones which displayed specific two-hybrid interactionswith both Skp1 and SIP-L. DNA sequencing analysis revealed 12 cDNAsencoding portions of SAF-1 and 9 cDNAs encoding portions of SAD. TheSAF-1 and SAD clones were sequenced and the resulting nucleotidesequences are set forth in SEQ ID Nos:7 (SAF-1α), 9 (SAF-1β), and 13(SAD).

Example 5 Isolation of Full-Length SAF-2 cDNAs

Full-length cDNA encoding a human SAF-2 protein was PCR-amplified fromZAPII Jurkat cDNA library (Stratagene) by using a forward primer5′-GTGAATTCATGCAACTTGTACCTGATATAGAGTTC-3′ (SEQ ID NO:44) containing anEcoRI site and a reverse primer 5′-GGACTCGAGGCTCTACAGAGGCC-3′ (SEQ IDNO:45), based on human DNA sequence from clone 341E18 on chromosome6p11.2-12.3 (AL031178). The PCR products were digested with EcoRI andXhoI, then directly subcloned into the EcoRI and XhoI sites of theplasmid pCDNA3. The corresponding plasmid was sequenced and the resultsare set forth in SEQ ID Nos: 11 and 12.

Example 6 Yeast Two-Hybrid Screen of SIP-L Binding Proteins

The human SIP-L cDNA encoding full-length SIP-L was cloned into thepGilda plasmid as a bait to screen a human embryonic brain cDNA library(Invitrogen) in EGY48 strain S. cerevisiae. From an initial screen of˜1.5×10⁷ transformants, 410 clones were identified that trans-activatedthe LEU2 reporter gene based on ability to grow on leucine-deficientmedia. Of those, 68 colonies were also positive for β-galactosidase.These 32 candidate transformants were then cured of the LexA/SIP-L baitplasmid by growth in media containing histidine and then mated with eachof 32 different indicator strains of cells containing one of followingLexA bait proteins: SIP-L, Bax (1-171), v-Ras, Fas (191-335), or BAG-1.The mating strain was RFY206 which had been transformed withpGilda-SIP-L or various control proteins and selected onhistidine-deficient media. This resulted in 16 clones which displayedspecific two-hybrid interactions with SIP-L. DNA sequencing analysisrevealed 3 cDNAs encoding portions of Skp1, 1 cDNA encoding portions ofSiah-1, and 11 cDNAs encoding portions of SIP-L. These results indicatethat SIP-L binds to Skp1 and Siah-1 proteins, and is able tohomodimerize with SIP isoforms.

Example 7 A Cell Proliferation Functional Assay of SIP/Siah Interaction

The effects of invention SIP-L and SIP-S proteins on Siah-1-induced cellcycle arrest in 293T epithelial cancer cells was examined and theresults are shown in FIG. 4. Human embryonic kidney 293 cells weremaintained in high-glucose DMEM medium containing 10% fetal calf serum,1 mM L-glutamine, and antibiotics. Cells (˜5×10⁵) in 60 mm plates weretransfected with a total of 3.0 μg of plasmid DNAs encoding Siah-1 aloneor together with SIP or SIP-S by a calcium phosphate precipitationtechnique. After 24 hours, the cells were harvested and the number ofviable and dead cells were counted using trypan blue dye exclusionassays. Efficiency of transient transfection was estimated by in situβ-galactosidase assay using a portion of the transfected cells. Thetransient transfection efficiency of the T293 cells was consistently90%.

As revealed in FIG. 4, over-expression of Siah-1 resulted in decreasednumbers of viable cells after 24 hours, without an increase in celldeath. Thus, Siah-1 suppresses proliferation of 293 cells.Co-transfection of SIP-L with Siah-1 did not substantially alterSiah-1-mediated growth suppression. In contrast, the SIP-S proteinabrogated the growth suppressive effects of Siah-1, which indicates thatthe invention SIP-S protein affects Siah-1 intracellularly in adifferent manner than SIP-L.

Example 8 In Vitro SIP: Siah-1 Protein Interaction Assays

Complementary cDNA encoding SIP-L was cloned into pGEX-4T-1 andexpressed in XL-1-blue cells (Stratagene, Inc.), and affinity-purifiedusing glutathione-Sepharose as is well-known in the art. PurifiedGST-fusion proteins (0.5-1.0 μg immobilized on 10-20 μl of glutathionebeads) and 2.5 ul of rat reticulocyte lysates (TNT-Lysates; Promega,Inc.) containing 35S-labeled in vitro translated (IVT) Siah-1 proteinswere incubated in 0.1 ml of HKMEN (10 mM HEPES [pH7.2], 142 mM KCl, 5 mMMgCl₂, 2 mM EGTA, 0.1% NP-40) at 4° C. for 30 minutes. The beads werewashed 3× with 1 ml HKMEN solution, followed by boiling in 25 μl ofLaemmli-SDS sample buffer. The eluted proteins were analyzed by SDS-PAGE(12%) and detected by fluorography. Use of equivalent amounts of intactGST-fusion proteins and successful IVT of each protein was confirmed bySDS-PAGE analysis using Coomassie staining or autoradiography,respectively.

The results are shown in FIG. 5A and indicate that Siah-1 binds to SIP-Land homodimerizes in vitro.

Example 9 Co-Immunoprecipitation Assay of SIP: Siah-1

Two×10⁶ 293T cells in 100 mm plates were transiently transfected with 10μg of pCDNA3-myc-SIP-L and 10 mg of pcDNA3-HA-Siah-1 (amino acids 97-298of SEQ ID NO:2). Twenty-four hours later, cells were disrupted bysonication in 1 ml of HKMEN solution containing 0.2% NP-40, 0.1 μM PMSF,5 μg/ml leupeptin, 1 μg/ml aprotinin, and 1 μg/ml pepstatin. Afterpreclearing with normal mouse IgG and 10 ml protein A-agarose,immunoprecipitations were performed using 10 ml of anti-mycantibody-conjugated sepharose (Santa Cruz) to precipitate the myc-SIP-Lfusion, or an anti-IgG as a control at 4° C. for 4 hours. Afterextensive washing in HKMEN solution, immune-complexes were analyzed bySDS-PAGE/immunoblotting using anti-HA antibody 12CA5 (BoehringerMannheim), followed by HRPase-conjugated goat anti mouse immunoglobulin(Amersham, Inc.), and detected using an enhanced chemiluminescence (ECL)system (Amersham, Inc.).

The results are shown in FIG. 5B and indicate that SIP proteins bind toSiah-1 intracellularly.

Example 10 Yeast Two-Hybrid Assay of Siah-1: APC Binding Specificity

One μg of plasmids encoding fusion proteins of the LexA DNA-bindingdomain fused to Siah-1, APC (2681-284), BAG-1, Bax, Ras, Fas, FLICE wereco-transformed into yeast strain EGY48 with 1 μg of pJG4-5 plasmidencoding fusion proteins of the B42 trans-activation domain fused to APC(2681-2843) and Siah-1. Transformed cells were grown on semi-solid medialacking leucine or containing leucine as a control which resulted inequivalent amounts of growth for all transformants. Plasmid combinationsthat resulted in growth on leucine-deficient media within 4 days werescored as positive (+). β-galactosidase activity of each colony wastested by filter assay and scored as blue (+) versus white (−) after 60minutes.

The results are shown in Table 1, and indicate that APC interactsspecifically by direct binding with Siah-1, and not with BAG-1, Bax,Ras, Fas nor FLICE.

TABLE 1 Specific Interaction of Siah with SIP Lex A B42 Leu⁺ β-Gal⁺Siah-1 APC (2681-2843) + + APC (2681-2843) Siah-1 + + BAG-1 APC(2681-2843) − − Bax APC (2681-2843) − − Ras APC (2681-2843) − − Fas APC(2681-2843) − − FLICE APC (2681-2843) − − empty APC (2681-2843) − −

Example 11 Yeast Two-Hybrid Assay of Siah-1: SIP Binding Specificity

One μg of plasmids encoding fusion proteins of the LexA DNA-bindingdomain fused to Siah-1, Siah-2, BAG-1, Bax, Ras, Fas, FLICE, and SIP-Lwere co-transformed into yeast strain EGY48 with 1 μg of pJG4-5 plasmidencoding fusion proteins of the B42 trans-activation domain fused toSIP-L, SIP-S, Siah-1, Siah-2, BAG-1, Bax, and Ras. Transformed cellswere grown on semi-solid media lacking leucine or containing leucine asa control which resulted in equivalent amounts of growth for alltransformants. Plasmid combinations that resulted in growth onleucine-deficient media within 4 days were scored as positive (+).β-galactosidase activity of each colony was tested by filter assay andscored as blue (+) versus white (−) after 60 minutes.

The results are shown in Table 2, and indicate that SIP proteinsinteract specifically by direct binding with Siah proteins. SIP-L wasfound to interact with Siah-1 and Siah-2, and not with BAG-1, Bax, Ras,Fas nor FLICE. SIP-S was also found to interact with Siah-1. Table 2also reveals that the SIP-L homodimerization domain is within aminoacids 73-228 of SIP-L (SEQ ID NO:4)

TABLE 2 Specific Interaction of Siah with SIP Lex A B42 Leu⁺ β-Gal⁺Siah-1 SIP-L + + Siah-1 SIP-S + + Siah-2 SIP-L + + BAG-1 SIP-L − − BaxSIP-L − − Ras SIP-L − − FLICE SIP-L − − empty SIP-L − − SIP-L Siah-1 + +SIP-L Siah-2 + + SIP-L BAG-1 − − SIP-L Bax − − SIP-L Ras − − SIP-LSIP-L + + SIP-L SIP-S − −

Example 12 Mapping of Siah-APC Interaction Domains

Expression plasmids encoding fusion proteins of Siah-1α fragmentscorresponding to: SEQ ID NO:2 amino acids 22-298; 22-251; 22-193;97-298; and 46-102, fused to the B-42 trans-activation domain wereco-transformed into yeast EGY48 cells with a plasmid encoding a chimericfusion protein of the Lex A DNA-binding domain fused to amino acids2681-2843 of APC “APC (2681-2843).” Transformed cells were grown onsemi-solid media lacking leucine or containing leucine as a control.Plasmid combinations that resulted in growth on leucine-deficient mediawithin 4 days were scored as positive (+). β-galactosidase activity foreach colony was tested by filter assay and scored as blue (+) versuswhite (−) (β-gal) based on a 1 hour of color development.

The results are shown in FIG. 3 and indicate that a region within the 47carboxy terminal amino acids of Siah-1α (SEQ ID NO:2) is required forbinding to APC.

Example 13 Mapping of SKP-1, SIP-L, SAF-1, and SAD Interaction Domains

Expression plasmids encoding fusion proteins of SAF-1α and functionalfragments thereof corresponding to SEQ ID NO:8 amino acids 68-443;80-443; and 258-443, were fused to the B-42 trans-activation domain.Likewise, expression plasmids encoding fusion proteins of SAD andfunctional fragments thereof corresponding to SEQ ID NO: 14 amino acids128-447; and 360-447, were fused to the B-42 trans-activation domain.These SAF-1-fragment- and SAD-fragment-B-42 fusion proteins wereco-transformed into yeast EGY48 cells with a plasmid encoding a chimericfusion protein of the Lex A DNA-binding domain fused to either SKP1,SIP-L, SAF-1, or SAD. Transformed cells were grown on semi-solid medialacking leucine or containing leucine as a control. Plasmid combinationsthat resulted in growth on leucine-deficient media within 4 days werescored as positive (+). β-galactosidase activity for each colony wastested by filter assay and scored as blue (+) versus white (−) (β-gal)based on a 1 hour of color development.

The results are shown in FIGS. 6A and 6B. FIG. 6A indicates that SAF-1interacts by direct binding to Skp1, SIP-L and SAD, but does notinteract with Siah-1. A region within the SAF-1 fragment correspondingto amino acids 80-257 of SEQ ID NO:8 is required for SIP-L interaction,whereas a region within amino acids 258-443 of SAF-1 is required forSkp1 and SAD interaction.

FIG. 6B indicates that SAD interacts by direct binding to Skp1, SIP-Land SAF-1, but does not interact with Siah-1. A region within the SADfragment corresponding to amino acids 1-127 of SEQ ID NO: 14 is requiredfor SAF-1 interaction; a region within amino acids 128-359 of SAD isrequired for Skp1 interaction; and a region within amino acids 360-447of SEQ ID NO: 14 is required for SIP-L interaction.

Example 14 Effect of Siah-1 Over-Expression on Stability of β-Catenin

293T cells were transiently transfected with a plasmid encodingmyc-tagged β-catenin and either pcDNA3, pcDNA3-Siah-1, or pcDNA3-Siah-1(97-298; amino acids 97-298 of SEQ ID NO:2). Whole cell lysates wereprepared, normalized for total protein content (25 μg per lane) andanalyzed by SDS-PAGE/immunoblotting using an anti-Myc tag antibody.

FIG. 7 indicates that expression of full-length Siah-1 abolishes, bydegradation, the presence of β-catenin within cells, whereas expressionof amino acids 97-298 of Siah-1 (SEQ ID NO:2) does not result inβ-catenin degradation. Thus, a region within amino acids 1-96 of SEQ IDNO:2 (Siah-1α), which contains the N-terminal “Ring” domain, is requiredfor protein degradation.

Example 15 Demonstration of SIP-Mediated Degradation of a TargetProtein, TRAF6

An invention SIP-based method for targeted degradation of proteins wasapplied to the degradation TRAF proteins. The schematic in FIG. 9 showsthe strategy employed for targeted degradation of specific TRAF-familyproteins. A chimeric protein is expressed from the plasmid pcDNA3 inwhich SIP-L (SEQ ID NO:3) is fused with bacterial thioredoxin containingvarious TRAF-binding peptides displayed on the surface of thioredoxin,as described by Brent and colleagues (Colas, et al. Nature, 380: 548,1996; Cohen, et al. Proc. Natl. Acad. Sci., 95: 14272, 1998; Geyer, etal. Proc. Natl. Acad. Sci., 96: 8562, 1999; Fabbrizio, et al. Oncogene,18: 4357, 1999). The TRAF-binding peptide binds to a member of theTRAF-family, and targets the TRAF-protein for ubiquitination andsubsequent proteosome-dependent degradation because the SIP-region ofthe chimeric protein recruits ubiquitin-conjugating enzymes (E2s) to theprotein complex.

Isolation of target-protein binding domain peptides that selectivelybind TRAF2 and TRAF6. A peptide aptamer library was screened by theyeast two-hybrid method to identify peptides that bind to either TRAF2or TRAF6 using the methods described in Leo, et al. J Biol Chem,274:22414, 1999. TRAFs are a family of signal transducing proteinsinvolved in cytokine receptor signaling inside cells. The sequences ofthe resulting TRAF-binding peptides are set forth in (Tables 3 and 4).

TABLE 3 Selected Traf 2 aptamer clones Clones (SEQ ID NO:)SLxCIxLR motif 219 (15) SESPGALRSGSLRCISLRIC 230 (16)VCRGRIRSGSLRCISLRICR 221 (17)   LLRLGCIRLLMLRRGVVFRL 208 (18)  VLFLSLRFWGLNIVVMGRLL 215 (19)    CRSLGVIVGGTEAAGAPTFI LS motif 208(20)    VLFLSLRFWGLNIVVMGRLL 213 (21) WLRRGLVGVFFLLSRVMVGI 218 (22)   SLGLSVCIGRRAGGGFRGFG 237 (23)    RFALSIGVCVVVRVGICLGM LV motif 209(24)    SAVLVLVYVSAALRGRGFGI 227 (25)   HGGGRGALVSVMYLCGFIRLNon-Consensus motif 231 (26)     RGRVIGMWVGLRCRMFLV

TABLE 4 Selected Traf 6 Aptamer Clones Clones (SEQ ID NO:) WR motif 625(27) VDWAVYSVVWRYTTT* 631 (28)  KTSVILVWRLSLFFCLYRSL* 606 (29)  ANRCWRE* 628 (30) EGTLSKRMWRTHN* 640 (31)    SWRDMTQSGM* 604 (32)  DVPWQRACARQ* 607 (33)  LERVARWVL* 602 (34)  VADVLVFWGYVF* DVxVF motif602 (34)  VADVLVFWGYVF* 613 (35)  GDVGVFPE* Non-Consensus motif 603 (36) PEMMLEGPKYCLxLxE* 609 (37)  LLYGALA* 612 (38)  GAIKFAHESCE* 616 (39) PMAMD* 632 (40)  QEEEM* 639 (41)  ISVVHGIGSDSD* *Termination codon

SIP-fusion chimeric protein construction. An invention SIP-fusionchimeric construct is generated by combining the open reading frame(ORF) of SIP_(L), followed immediately by restriction enzyme sitesallowing for subcloning of desired target-protein-binding domains (e.g.peptides or protein domains). These SIP-fusions are then transfectedinto mammalian cells to eliminate by protein degradation specific targetproteins which bind the subcloned peptides/protein domains by recruitingthem into the ubiquitin conjugating complex.

Engineering of the parent SIP-vector (SIPpcDNA3.1) cassette.Oligonucleotides corresponding to the 5′ and 3′end of SIP_(L) were usedin PCR to amplify the entire ORF of SIP_(L) (SEQ ID NO:3). The forwardprimer contains a Hind III restriction site linker(5′-GATCAAGCTTATGGCTTCAGAAGAGCTACAG; (SEQ ID NO:46) restriction site isunderlined) followed immediately by the SIP_(L) (SEQ ID NO:3) startcodon; the reverse primer contains an EcoRI restriction site andmutations in the stop codon allowing for translational readthrough(5′-GATCGAATTCtccAAATTCCGTGTCTCCTTTGGCTTG; (SEQ ID NO:47) mutated stopcodon is in lowercase). The generated PCR product was then agarosegel-purified and digested with Hind III and EcoRI restriction enzymes(New England BioLabs; Beverly, Mass.). The product was againgel-purified before ligating into Hind III/EcoRI digested pcDNA3.1expression vector (Invitrogen; Carlsbad, Calif.) with T4-DNA ligase (NewEngland BioLabs). This construct was termed SIPpcDNA3.1.

For the construction of SIP-thioredoxin (Trx) peptide-aptamer fusions,clones from a peptide-aptamer library screened against Traf6 (see Table4) were amplified by PCR with the following primers:

Forward: (SEQ ID NO: 48) 5′-CCTCTGAATTCCATATGAGCGATAAAATTATTCACC;(EcoRI underlined) Reverse: (SEQ ID NO: 49)5′-GATCCTCGAGTAGATGGCCAGCTAGGCCAGGTTA. (Xho I underlined)

The resulting PCR products (˜350-370 bp) contain the ORF of thioredoxin(Trx) with the selected peptide aptamers inserted into its active-loop.The products were then digested with EcoRI and Xho I before ligatinginto the EcoRI/XhoI-digested SIPpcDNA3.1 cassette using T4-DNA ligase.Final clone constructs were numbered and were confirmed by sequencingbefore using in transfection studies.

Tranfection: HEK293T cells were transiently transfected by alipofectamine method with various amounts (1 vs 4 μg) of pcDNA3 plasmidsencoding either SIP-TR fusion protein lacking a TRAF6-binding peptide(“SIP”) or SIP-TR fusion protein displaying one of the peptides shown inTable 4 above (set forth in FIG. 10 as S603, S604, S606). In some cases,the proteosome inhibitor MG132 (10 μM) was added to cultures to preventprotein turnover. SIP* in FIG. 10 corresponds to the control expressionproduct of parental construct SIP pcDNA3.1

To determine the efficacy of the SIP:TRAF-binding peptide chimericproteins, levels of TRAF6 protein were then measured two days later byimmunoblotting using a anti-TRAF6-specific antiserum (Santa CruzBiotech, Inc.) in experiments where HEK293T cell lysates were normalizedfor total protein content (25 μg per lane). The cell lysates wereanalyzed by SDS-PAGE/immunoblotting using an enhanced chemiluminescencedetection method, as described previously (Leo, et al. J Biol Chem, 274:22414, 1999). The results shown in the left panel of FIG. 10 show thatSIP-TR fusion proteins displaying TRAF6-binding peptides (S603, S604,and S613) induce a reduction in TRAF6 protein levels, with the S603peptide representing the most potent of these.

To determine the specificity of the SIP:TRAF-binding peptide chimericproteins, the same immunoblots were reprobed with an antiserum againstSIP to demonstrate equivalent levels of production of SIP-TR fusionproteins, or with antibodies specific for TRAF2 to reveal selectivedegradation of TRAF6 but not TRAF2. The results shown in the right panelof FIG. 10 show that addition of a proteosome inhibitor, MG132, preventsthe reductions in TRAF6. Note also that TRAF2 protein is not degraded,demonstrating the specificity of the targeting approach.

Example 16 Demonstration of Ornithine decarboxylase-(ODC) MediatedDegradation of a Target Protein

The levels of intracellular proteins are regulated byproteasome-dependent proteolysis. The selective degradation of cellularproteins is mediated primarily by both the ubiquitin-dependent and-independent proteasome pathways. In this example, the substratereceptor of a major proteolytic machinery is engineered to direct thedegradation of otherwise stable cellular proteins in mammalian cells.

Strategy for the selective degradation of cellular protein by chimericODC proteins and antizyme. Ornithine decarboxylase (ODC) is degraded ina 26S proteasome antizyme-dependent manner, which does not requireubiquitination. In order to target the protein of interest (Targets),the adapter protein or peptide, which binds to target protein, iscovalently fused to ODC for the degradation of protein complex by the26S proteasome. A model of antizyme-dependent targeted proteindegradation by ODC-conjugated proteins is shown in FIG. 11A. FIG. 11Bshows the amino acid sequence of the ODC-fusion protein (SEQ ID NO:50)(human ODC sequence GenBank M16650).

Targeted degradation of TRAF6 by ODC-TRAF6C and ODC-RANK peptide. Fortargeted degradation of TRAF6, HEK293T cells were transientlytransfected with plasmid encoding HA-TRAF6 (0.5 μg), HA-TRAF2 (0.5 μg),ODC (0.5 μg), ODC-TRAF6C (0.5 μg), ODC-RANK peptide (0.5 μg) ODC-CD40CT(0.5 μg) or myc-Antizyme (0.5 μg) in various combinations, as indicatedin FIG. 12 (total DNA amount normalized). After 24 h, cell lysates wereprepared from duplicate dishes of transfectants, normalized for totalprotein content (20 μg per lane), and analyzed by SDS-PAGE.Immunoblotting was performed using antibodies specific for the tags HA(TRAF6) or Myc (ODC or Antizyme), with detection by enhancedchemiluminescence (ECL). Levels of TRAF6 mRNA were measured by Northernblot (mRNA).

Expression of ODC-TRAF6C in HEK293T cells induced marked reductions inHA-TRAF proteins, with or without co-expressing plasmids encodingantizyme. In contrast, expression of ODC-TRAF6-RANK peptide decreasedTRAF6 proteins in an antizyme-dependent manner. Expression of ODC-CD40did not reduce TRAF6 protein. Neither ODC-TRAF6C nor ODC-RANK peptideaffected the expression of TRAF2 proteins. Moreover, reductions in TRAF6protein levels were not due to a decrease in TRAF6 mRNA, as determinedby northern blot. Therefore, the degradation of TRAF6 by ODC-TRAF6C wasantizyme-independent but the degradation of TRAF6 by ODC-RANK peptidewas antizyme-dependent.

Other examples for ODC-adapter-induced degradation of target protein(s).The activity of ODC-adapter-induced degradation of other targetprotein(s) was tested. Antizyme-dependent targeted degradation ofretinoblastoma (Rb) by ODC-E7 peptide is shown in FIG. 13A. HEK293Tcells were transiently transfected with plasmid encoding HA-Rb (0.5 μg),ODC (0.5 μg), ODC-E7 peptide (0.5 μg) or myc-Antizyme (0.5 μg) invarious combinations, as indicated in FIG. 13A (total DNA amountnormalized). Antizyme-independent targeted degradation of Cdk2 byODC-p21waf-1 is shown in FIG. 13B. HEK293T cells were transientlytransfected with plasmid encoding myc-Cdk2 (0.5 μg), ODC (0.5 μg),ODC-p21waf-1 (0.5 μg) or myc-Antizyme (0.5 μg) in various combinations,as indicated in FIG. 13B (total DNA amount normalized).Antizyme-independent targeted degradation of IKKβ by ODC-IKKβ(leucine-zipper domain) is shown in FIG. 13C. HEK293T cells weretransiently transfected with plasmid encoding HA-IKKβ (0.5 μg), ODC (0.5μg), ODC-IKKβ-LZ(0.5 μg) or myc-Antizyme (0.5 μg) in variouscombinations, as indicated in FIG. 13C (total DNA amount normalized).After 24 h, cell lysates were prepared from duplicate dishes oftransfectants, normalized for total protein content (20 μg per lane),and analyzed by SDS-PAGE. Immunoblotting was performed using antibodiesspecific for Rb, Cdk2, IKKβ or HSC70 (as a control), with ECL-baseddetection.

Analysis of interactions of ODC-TRAF6C and TRAF6. For analysis ofinteractions of ODC-TRAF6C and TRAF6, HEK293T cells were transientlytransfected with plasmids encoding hemaglutinin (HA)-tagged TRAF6 andODC (0.5 μg), ODC-TRAF6C peptide (0.5 μg) or antizyme (0.5 μg) invarious combinations, as indicated in FIG. 14 (total DNA amountnormalized). After 24 h, 10 μM MG132 was added into culture media, andthe cells were incubated another 6 hours. Lysates were normalized fortotal protein content and subjected to immunoprecipitation using 20 μlof anti-myc monoclonal antibody-conjugated beads. After recoveringimmune-complexes with beads and washing, the immunoprecipitates wereanalyzed by SDS-PAGE. Immunoblotting was performed using an anti-HAmonoclonal antibody, with ECL-based detection. As a control, 0.1 volumeof input cell lysate was loaded directly in the same gel.

To preliminarily assess whether ODC-TRAF6 and antizyme can exist in acomplex with TRAF6, the TRAF6 proteins were tested forco-immunoprecipitation with ODC-TRAF6C using monoclonal antibodiesagainst the myc epitope tag. As shown in FIG. 14, TRAF6 was recovered inTRAF6C-ODC, but not control ODC-empty, immune-complexes prepared fromcells expressing ODC-TRAF6C.

Targeted degradation of TRAF6 by ODC-RANK peptide isproteasome-dependent. To test whether targeted degradation of TRAF6 byODC-RANK peptide is proteasome-dependent, HEK293T cells were transientlytransfected with 0.2 μg plasmid encoding HA-TRAF6 (0.5 μg), ODC-RANKpeptide (0.5 μg) or myc-Antizyme (0.5 μg) in various combinations, asindicated in FIG. 15 (total DNA amount normalized). After 24 h, cellswere either untreated or treated with 1 μM MG132 (MG132), 1 nMEpoximycine (Epox.), 10 μM Lactastacine (Lact.) or 1 μM Trypsininhibitor (Tryp.) for 6 hours. Cell lysates were prepared from duplicatedishes of transfectants, normalized for total protein content (20 μg perlane), and analyzed by SDS-PAGE. Immunoblotting was performed usingantibodies specific for HA.

As shown in FIG. 15, degradation of TRAF6 by ODC-RANK peptide andantizyme was inhibited by proteasome inhibitors, MG132, Epoximycine andLactastacine but not by trypsin inhibitor. These results indicate thatthe degradation is S26 proteasome dependent.

Pulse-chase analysis of TRAF6 turnover rate. Pulse-chase analysis ofectopically expressed HA-tagged TRAF6 was performed. HEK293T cells weretransiently co-transfected with plasmids encoding HA-TRAF6 and ODC-RANKpeptide, with or without myc-Antizyme. After 24 hours, cells werepulse-labeled with ³⁵S-methionine and cysteine, and then chased withmedia lacking the labeled amino acids. Cells were lysed at the indicatedtimes (FIG. 16), and the expressed HA-TRAF6 was recovered byimmunoprecipitation via a HA epitope tag. Immunoprecipitad HA-TRAF6 wassubjected to SDS-PAGE, and dried gels were analyzed with aPhosphorImager. Data from pulse-chase analysis is presented as theaverage±SD from duplicate experiments (FIG. 16B; −antizyme, closedcircles; +antizyme, open circles). The blots shown are representative ofduplicate experiments (FIG. 16A).

The turnover rate of the TRAF6 was increased in the 293T cellstransiently transfected with ODC-RANK peptide and antizyme, as comparedto the cells tranfected with ODC-RANK peptide alone. These resultsdemonstrate that ODC-RANK peptide and antizyme promote down-regulationof TRAF6 in a post-translational manner.

Functional analysis using ODC-E7 peptide. To assess whether ODC-E7peptide reduces endogenous Rb protein level and affects its cellularfunction, HEK293T cells were transiently transfected with ODC-E7 peptideand antizyme. Degradation of endogenous Rb protein by ODC-E7 is shown inFIG. 17A. HEK293T cells (100 mm dish) were transiently transfected with2 μg plasmid encoding ODC (2 μg), ODC-E7 peptide (2 μg) or myc-Antizyme(2 μg) in various combinations, as indicated in FIG. 17A (total DNAamount normalized). After 48 h, lysates were normalized for totalprotein content and subjected to immunoprecipitation using 1 μg ofanti-Rb monoclonal antibody. After recovering immune-complexes withprotein G and washing, the immunoprecipitates were analyzed by SDS-PAGE.Immunoblotting was performed using an anti-Rb monoclonal antibody withECL-based detection.

The effect of ODC-E7 on E2F reporter activity was also tested. HEK293Tcells were transiently transfected with a reporter gene plasmid (0.1 μg)that contains a E2F responsive element cloned upstream of a luciferasereporter gene, together with 0.01 μg of pCMVβ-gal as atransfection-efficiency control, and 0.1 μg of the indicated plasmidsencoding ODC, ODC-E7 or Antizyme in various combinations, as indicatedin FIG. 17B (bars correspond to plasmid combinations as indicated inFIG. 17A) (total DNA amount normalized). Luciferase activity wasmeasured in cell lysates 24 hr later and normalized relative toβ-galactosidase (mean±std. dev.; n=3).

As shown in FIG. 17A, endogenous Rb protein was degraded by ODC-E7peptide with antizyme. Since transcription factor E2F activity isnormally suppressed by Rb in proliferating cells, the effects of ODC-E7and antizyme on E2F activity was explored using transient transfectionreporter gene assays. Expression of ODC-E7 plus antizyme induceda >5-fold increase in E2F transcriptional activity in HEK293T cell lines(FIG. 17B). In contrast, ODC-empty plus antizyme or ODC-E7 alone failedto activate E2F transcription activity. Therefore, ODC-E7 peptideaffects endogenous Rb protein levels and E2F activity.

Effect of ODC-RANK peptide and ODC-TRAF6C on IL-1-induced NFκB reporteractivity. Previous data have suggested that TRAF6 may play an importantrole in IL-1-mediated NFκB activation but not in TNFα-mediated NFκBactivation. Therefore, the effects of ODC-TRAF6C and ODC-RANK peptide onboth IL-1- and TNFα-induced NFκB activation were examined.

To test the effect of ODC-RANK peptide and ODC-TRAF6C on IL-1-inducedNFκB reporter activity, HEK293T cells were transiently transfected witha reporter gene plasmid (0.1 μg) that contains a NFκB responsive elementcloned upstream of a luciferase reporter gene, together with 0.01 μg ofpCMVβ-gal as a transfection-efficiency control, and 0.1 μg of plasmidsencoding ODC, ODC-TRAF6C, ODC-RANK peptide, or Antizyme in variouscombinations, as indicated in FIG. 18 (total DNA amount normalized).After 24 hours, cells were treated with 50 ng/ml IL-1 or 10 ng/ml TNFαfor an additional 24 hours. Luciferase activity was measured in celllysates and normalized relative to β-galactosidase (mean±std. dev.;n=3).

As shown in FIG. 18, expression of ODC-TRAF6C or ODC-RANK peptide inHEK293T cells induced marked reductions in NFκB reporter activity. Incontrast, ODC-TRAF6C and ODC-RANK peptide did not affect theTNFα-madiated NFκB activation. These results confirm that TRAF6 is animportant mediator in IL-1-induced signal transduction by usingconditional inactivation of TRAF6 proteins.

A summary of the activity of various protein-degradation binding domainsfused with ligands (target-protein binding domains) in the degradationof particular targets is shown in Table 5.

Example 17 Targeting Protein Destruction by using aUbiquitin-Independent, Proteasome-Mediated Degradation Pathway

Plasmids. The cDNAs encoding human AZ (70-228) and human ODC(full-length) were PCR-amplified from either human placenta or humanfetal brain randomly primed cDNA libraries (Stratagene). To create theC-terminal fusion cassette vectors, cDNAs encoding ODC (full-length),Siah-interacting protein (SIP) (full-length), Siah1 (full-length),monoubiquitin (full-length), and S5a (full-length) were PCR-amplifiedwith a forward primer containing BglII site on the 5′ end and a reverseprimer containing EcoRI site on the 3′ end and subcloned into the BamHIand EcoRI sites of pcDNA3 plasmid (Invitrogen) with an N-terminal Mycepitope-tag (MEQKLISEEDL) and flexible linkers (FLs) consisting ofeither the Bcl-2 loop (residues 31 to 94), GGS, or AGGGSGGGGSGGGGSGGGGS.To create amino-terminal fusion cassette vectors, cDNAs encoding E7(31-105) and Fbx7 (full-length) were PCR-amplified with a forward primercontaining a XhoI site on the 5′ end and a reverse primer containingApaI site on the 3′ end and subcloned into the XhoI and ApaI sites ofthe pcDNA3-myc vector. Ubiquitin mutant (G76V) was generated by two-stepPCR-based mutagenesis using a full-length human monoubiquitin cDNA.Adaptors, TRAF6 (271-530), TRAF2 (ARING), E7 (2-34), p21^(waf-1)(full-length), Caspase-9 (1-92), Apaf-1 (1-87), FADD (1-78), IKKα(304-758), IKKβ (305-745), and BAG-1 (full-length), were PCR-amplifiedand subcloned into each of the cassette vectors. To create theluciferase reporter plasmid containing an E2F binding site, primers5′-CTGCAATTTCGCGCCAAACTTGTGCAATTTCGCGCCAAACTTGC-3′ and5′-TCGAGCAAGTTTGGCGCGAAATTGCACAAGTTTGGC GCGAAATTGCACTCGA-3′ wereannealed and ligated into pGL3E vector cleaved with SacI and HindIII(Krek et al., Science 262:1557-1560, 1993). The reporter gene plasmidcontaining four tandem HIV-NFκB response elements has been described inGalang et al., Oncogene 9:2913-2921, 1994.

Transfections and Cell Culture. HEK293T cells were maintained inhigh-glucose DMEM containing 10% FCS, 1 mM L-glutamine, and antibiotics.Cells (approximately 5×10⁵) in six-well plates were transfected withplasmid DNAs by using Lipofectamine 2000 (Invitrogen). In some cases, 50μg/ml cycloheximide was added to prevent polypeptide synthesis.

Immunoblots. Cells were lysed in 1 ml of HKMEN solution containing 0.5%Nonidet P-40, 0.1 μM PMSF, 5 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/mlpepstatin, and 20 μM MG132. Lysates were analyzed bySDS/PAGE/immunoblotting using various antibodies, followed byHRPase-conjugated goat anti-mouse or anti-rabbit Ig (Amersham Pharmacia)and detected by using enhanced chemiluminescence (Amersham Pharmacia).

Pulse-Chase Analysis. For pulse-chase analysis of ectopically expressedHA-tagged TRAF6 (271-530) or IKKβ (305-745), HEK239T cells weretransiently transfected in six-well plates. After 24 h, cells werepulse-labeled for 1 h with 0.1 mCi (1 Ci=37 GBq) of [³⁵S]methionine and[³⁵S]cysteine per well and then chased with cold media. Cells were lysedin RIPA buffer (0.05 M Tris·HCl, pH 7.2/0.15 M NaCl/1% Triton X-100/1%deoxycholate/0.1% SDS) supplemented with protease inhibitors. Afterpreclearing with 20 μl of protein G-Sepharose for 1 h at 4° C., HA-TRAF6or HA-IKKβ were immunoprecipitated by using rat anti-HA monoclonalantibody (3F10, Invitrogen) adsorbed to protein G-Sepharose beads at 4°C. for 4 h. After three washes with RIPA buffer, immunoprecipitates weresubjected to SDS/PAGE. Dried gels were analyzed with a PhosphorImager(Molecular Dynamics).

Reporter Gene Assays. NFκB and E2F transcriptional activity weremeasured in HEK293T cells by transient transfection reporter geneassays. Cells at approximately 50% confluence in 24-well plates werecotransfected with 0.1 μg of reporter plasmids containing NFκB- orE2F-binding sites cloned upstream of a luciferase gene, 0.01 μg ofpCMVP-LacZ control plasmid, and 0.1 μg of various additional plasmids,as indicated. After 24 h, cells were lysed, and the relative amount ofluciferase activity was measured according to the manufacturer'sinstructions (Promega), normalizing all values relative tobeta-galactosidase activity.

Results

To explore technologies for inducing proteasome-dependent degradation oftarget polypeptides, we engineered plasmids to express in mammaliancells various chimeric polypeptides in which a polypeptide involved inubiquitination mechanisms was fused to polypeptides or protein domainsknown to interact with specific target polypeptides. Aprotein-interaction domain (adapter) is expressed with ODC attached toits C terminus with a flexible linker (FL) sequence AGGGS(GGGGS)₃. ODCis known to form homodimers. The ODC-adapter fusion binds targetpolypeptides and docks on the 26S proteasome. AZ catalyzes degradationof the ODC-adapter polypeptide by the proteasome along with theinteracting target polypeptide.

Twelve pairs of interacting polypeptides were studied, chosen randomlyfrom reagents available in our laboratory, including: (i) the C-terminalTRAF domain (residues 271-530) of the adapter protein TRAF6, which bindsTRAF6 (Ishida et al., J. Biol. Chem. 271:28745-28748, 1996), (ii) apeptide (residues 341-349) from the cytosolic domain of the TNF receptor(TNFR)-family protein RANK, known to bind TRAF6 (Ye et al., Nature418:443-447, 2002); (iii) the cytosolic domain of TNFR-family memberCD40, known to bind TRAF2 (Rothe et al., Science 269:32767-32770, 1995),(iv) I-TRAF, a TRAF2-binding protein (Rothe et al., Proc. Natl. Acad.Sci. USA 93:8241-8246, 1996); (v and vi) the leucine zipper of IKKβ,which binds IKKα and IKKβ (Woronicz et al., Science 278:866-869, 1997);(vii) a peptide from papillomavirus E7 protein, which binds Rb; (viii)the CARD domain of proCaspase-9, which binds Apaf1; (ix) The CARD domainof Apaf-1, which binds proCaspase-9 (Zhou et al., Proc. Natl. Acad. Sci.USA 96:11265-11270, 1999); (x) the DED domain of adapter protein FADD,which binds proCaspase-8; (xi) BAG1, a Hsp70-binding cochaperone(Takayama et al., EMBO J. 16:4887-4896, 1997), and (xii) p21^(Waf-1), aCdk2 inhibitor (Harper et al., Cell 75, 805-816, 1993). Protein orpeptide ligands were expressed in HEK293T cells as fusion polypeptideswith an N- or C-terminus-appended protein known to participate inprotein-ubiquitination reactions, including (i) SIP, a protein known tobind the E3 ligase Siah1 and the Skp1 protein of Skp1/cullin/F-boxprotein complexes (Matsuzawa and Reed, Mol. Cell 7:915-926, 2001); (ii)Siah-1, a RING-domain containing E3 ligase (Matsuzawa et al., EMBO J.17:2736-2747, 1998); (iii) E7, a papillomavirus protein with reported E3ligase activity (Boyer et al., Cancer Res. 56:4620-4624, 1996; Jones etal., Virology 258:406-414, 1999); (iv) a fragment of the F-box proteinFbx7, in which the substrate-binding domain was substituted, leaving theSkp1-binding region; (v) ubiquitin G76V, a nonhydrolyzable variant ofubiquitin previously used to confer proteasome-sensitivity onpolypeptides; (vi) a tandem 4′ oligomer of ubiquitin G76V (Stack et al.,Nat. Biotechnol. 18:1298-1302, 2000); and (vii) S5a, a subunit of theproteasome involved in substrate recognition (Deveraux et al., J. Biol.Chem. 270:23726-23729, 1995). In most cases, the protein ligand wasseparated from the ubiquitin-pathway domain by a FL consisting of thesequence AGGGS(GGGGS)₃ (Hoedemaeker et al., J. Biol. Chem.272:29784-29789, 1997). All constructs included an epitope tag, allowingverification of polypeptide production by immunoblotting andconfirmation of binding to cellular target polypeptides byco-immunoprecipitation assays.

The ability of these fusion polypeptides to induce reductions in thesteady-state levels of endogenous and plasmid-expressed interactingproteins was then explored by immunoblot analysis of HEK293T cellstransfected to high efficiency (>90%) with plasmids encoding the fusionpolypeptides. The target polypeptides were coexpressed with epitope tagsby cotransfection by using a plasmid with a strong constitutive promoter(CMV immediate-early region promoter) to ensure continuous production oftarget polypeptides and avoid artifactual reductions that might becaused by unanticipated effects of the chimeric fusion polypeptides onpathways that affect endogenous gene expression and to avoidfalse-negatives due to nontransfected cells. Although not every possiblecombination was tested (Table 6), none of these fusion polypeptidessuccessfully reduced levels of target polypeptides, based on 40combinations tested.

TABLE 6 Ligand Target ODC (N) ODC + Az SIP (N) Siah (N) E7 (C) Fbx7 (C)Ub1 (N) Ub4 (N) S5a (N) TRFAF6-C TRAF6 ↓ ↓ — — — — — — — RANK-pep. TRAF6— ↓ — — — — — — — CD40CT TRAF2 . . . . . . nd nd nd nd nd nd . . .I-TRAF TRAF2 . . . . . . . . . nd nd nd nd nd . . . IKKα(LZ) IKKα . . .. . . . . . nd nd nd nd nd . . . IKKβ(LZ) IKKβ ↓ ↓ . . . nd nd nd nd nd. . . E7 Rb . . . ↓ . . . . . . nd . . . . . . . . . . . .Caspase9(CARD) Apaf1 . . . . . . . . . . . . . . . . . . . . . . . . ndApaf1(CARD) Caspase9 nd . . . . . . . . . . . . . . . . . . . . . ndFADD(DED) Caspase8 nd — — — — — — — nd BAG-1 ESP70 — — nd nd nd nd nd ndnd p21 Cdk2 ↓ ↓ nd . . . . . . nd nd nd nd Success Ratio All Targets5/12 0/9 0/7 0/6 0/6 0/6 0/6 0/7

Testing of an AZ-Based System for Targeted Polypeptide Degradation.Given the lack of efficacy of fusion polypeptides based on knowncomponents of the ubiquitination machinery to effectively inducedegradation of interacting polypeptides, we turn to an alternativestrategy based on knowledge of the mechanism by which ODC is degraded bythe proteasome through ubiquitin-independent mechanisms. For thispurpose, protein ligands were expressed as fusion polypeptides with ODCat their C termini, thus exposing the C terminus of ODC, which is knownto bind the proteasome independently of ubiquitin.

An FL sequence was also inserted between the protein ligands and ODC.Three types of linkers were tested, including a 63-aa segment from theBcl-2 protein (residues 31-94), which is known from structural studiesto constitute a nonstructured, flexible peptide rich in prolines andglycines (Chang et al., EMBO J. 16:968-977, 1997), a Gly-Gly-Protripeptide, and the sequence [Gly-Gly-Gly-Gly-Ser]₃. These ODC fusionpolypeptides were then expressed in HEK293T cells alone or incombination with AZ, which binds ODC and catalyzes its degradation bythe 26S proteasome (Murakami et al., Biochem. Biophys. Res. Commun.267:1-6, 2000). Again, polypeptide targets were coexpressed fromplasmids with epitope tags for initial experiments, and their levels ofexpression were evaluated by SDS/PAGE/immunoblotting.

Of the twelve pairs of protein interactions tested by using the ODC/AZsystem, five resulted in specific reductions of the target polypeptide(Table 1). For two of these five successful knock-downs, reductions oftarget polypeptide were entirely dependent on co-expression of AZ withthe ODC chimeric fusion polypeptide, whereas, in another case, AZenhanced the reduction caused by the ODC chimeric fusion alone. In theother two successful cases, the ODC chimeric fusion polypeptide wassufficient by itself, suggesting that fusing certain polypeptides to ODCmay supplant the need for AZ. In this regard, the AZ polypeptide isknown to induce a conformational change in ODC that exposes aproteasome-binding domain in its C terminus (Murakami et al., Biochem.Biophys. Res. Commun. 267: 1-6, 2000), raising the possibility that somefusion partners mimic this effect of AZ.

We empirically determined that inclusion of a FL between ODC and theprotein-interaction domain can be critical for successful degradation ofcellular substrates, with the [Gly-Gly-Gly-Gly-Ser]₃ linker yieldingbest results. FIG. 19 provides an example, comparing the efficiency of aODC fusion containing a Rb-binding fragment (amino acids 2-34) expressedwith or without a FL, with respect to degradation of cellular target Rb.

Analysis of Specificity of AZ-Based Polypeptide Target Degradation. Weperformed a variety of experiments to explore the specificity of theODC/AZ system for inducing target polypeptide reductions. For example,the target polypeptide TRAF6 is a member of a family of six mammalianadapter proteins with differential specificity for peptidyl motifslocated in the cytosolic domains of TNF-family receptors (Ishida et al.,J. Biol. Chem. 271:28745-28748, 1996). A C-terminal region in thesepolypeptides (C-TRAF domain) is responsible for TNFR binding. Theseadapter proteins also form homotrimers through the proximal portion oftheir TRAF domains (Arch et al., Genes Dev. 12:2821-2830, 1998). Wetherefore contrasted the levels of TRAF6 and TRAF2 polypeptides in cellsexpressing ODC chimeric fusion polypeptides containing either the TRAFdomain of TRAF6 or a TRAF6-binding peptidyl motif from the cytosolicdomain of RANK (“RANKp”). ODC nonfusion polypeptide served as a negativecontrol. FIG. 20 shows the selective degradation of TRAF6 but not TRAF2by ODC chimeric fusion polypeptides. As shown in FIG. 20, co-expressingODC-C-TRAF6 caused reductions in the levels HA-TRAF6 but not HA-TRAF2polypeptide. Cotransfection of AZ further decreased levels of HA-TRAF6but not HA-TRAF2. Levels of HA-TRAF6 were reduced in cells expressingODC-RANKp only when AZ was coexpressed. ODC-RANKp did not affect levelsof HA-TRAF2, confirming the specificity of these results. ODC controlpolypeptide also did not alter levels of HA-TRAF6 or HA-TRAF2.

Immunoblot analysis confirmed production of the ODC-chimeric fusionpolypeptides and AZ in the transfected cells. Note that accumulation ofODC-C-TRAF6 was markedly reduced, compared with ODC-RANKp, suggestingthat fusing C-TRAF6 to ODC promotes its proteasome-dependent degradationindependent of AZ. As expected, reductions in ODC-RANKp were induced bycoexpressing AZ, consistent with AZ-dependent degradation of this ODCchimeric fusion polypeptide. Thus, we surmise that some ODC chimericfusion polypeptides spontaneously associate with and are efficientlydegraded by the 26S proteasome (e.g., ODC-C-TRAF6), whereas others(e.g., ODC-RANKP) require AZ as a cofactor for their degradation, likewild-type ODC.

AZ/ODC System Increases Rate of Target Polypeptide Degradation. Next, weundertook experiments to determine the mechanism by which targetpolypeptide reductions were achieved when using the ODC/AZ system. FIG.21 shows the AZ-dependent, proteasome-dependent degradation of targetpolypeptide induced by ODC chimeric fusion polypeptides. First, wedetermined the effect of ODC chimeric fusion polypeptides on the levelof mRNA encoding target polypeptides, anticipating that mRNA levelsshould be unchanged. Analysis of TRAF6 mRNA levels in cells transfectedwith plasmids encoding AZ and either ODC-C-TRAF6 or ODC-RANKp fusionpolypeptides confirmed no effect on expression at the mRNA level (FIG.20). Second, we explored the effects of pharmacological inhibitors ofthe 26S proteasome. FIG. 21A shows an example where HEK293T cells werecotransfected with a plasmid encoding HA-TRAF6 alone or in combinationwith plasmids encoding AZ and an ODC chimeric fusion polypeptidecontaining a TRAF6-binding peptide from the cytsolic domain of RANK(RANKp). Coexpression of ODC-RANKp and AZ with HA-TRAF6 resulted inprofound reductions in the steady-state levels of HA-TRAF6 polypeptide,as determined by immunoblotting. Culturing these transfected cells withproteasome inhibitors MG132, epoximycin, or lactacystin restoredHA-TRAF6 levels. In contrast, a trypsin inhibitor, used here as acontrol, was ineffective (FIG. 21A). These data demonstrate thatODC/AZ-induced degradation of target polypeptides isproteasome-dependent. Third, we determined the effects of the ODC/AZsystem on protein half-life using ³⁵S-L-methionine pulse-chase methods.FIG. 21B shows results comparing the half-life of HA-TRAF6 in cellsco-transfected with ODC-RANKp, with or without AZ. In cells expressingAZ, the half-life of HA-TRAF6 was reduced from approximately 2 h to <1h, consistent with target polypeptide degradation occurring via anAZ-dependent mechanism. We conclude, therefore, that the ODC/AZ systeminduced proteasome-dependent degradation of target polypeptides withoutaffecting mRNA expression. Pulse-chase experiments were also performedfor IKKβ, comparing cells transfected with plasmids encoding ODC versusODC-IKKβ. The starting levels of IKKβ were lower in cells expressingODC-IKKβ before initiating the chase, suggesting ongoing degradation.Cold L-methionine chase revealed that, indeed, the rate of degradationof IKKβ was faster in cells expressing ODC-IKKβ, compared with ODCcontrol (FIG. 21C). Fourth, we also used another approach to gauge therates of target-polypeptide degradation where cells were transfectedwith ODC-expressing plasmids and then protein synthesis was shut off aday later by adding cycloheximide to cultures. Using ODC-p21 as anexample, we compared the rate of degradation of the p21 targetFlag-tagged Cdk2 in HEK293T cells transfected with ODC-control orODC-p21 plasmids. Before cycloheximide treatment, steady-state levels ofFlag-tagged Cdk2 were lower in the ODC-p21-expressing cells comparedwith ODC-control cells (FIG. 21D), suggesting ongoing degradation. Afteraddition of cycloheximide, the rate of decline in Cdk2 polypeptidelevels was faster in ODC-p21-expressing cells, as determined bydensitometric quantification of immunoblot data developed by using ananti-Flag antibody with chemiluminescent detection.

Modulating Cellular Pathways by Using AZ-Based Targeted PolypeptideDegradation. Finally, we explored whether the ODC/AZ system could beused to successfully ablate the function of endogenous polypeptides.First, we examined the effects of ODC-C-TRAF6 and ODC-RANKp on inductionof NFκB activity in HEK293T cells exposed to either TRAF6-dependent(e.g., IL-1) or independent (e.g., TNFα) cytokines. FIG. 22 shows thefunctional ablation of endogenous TRAF6 by ODC/AZ system. IL-1 inducedmarked increases in NFκB activity in HEK293T cells, as determined byreporter gene assays, which were reduced to near baseline levels byexpression of ODC-C-TRAF6 or by coexpression of ODC-RANKp with AZ. Asexpected, ODC control polypeptide had no effect on NFκB induction byIL-1, confirming the specificity of these results. In contrast to theeffects of ODC-C-TRAF6 and ODC-RANKp chimeric fusion polypeptides onIL-1 signaling, induction of NFκB activity by TNFα was unimpaired,consistent with the differential use of TRAF-family adapter proteins byIL-1 (e.g., TRAF6) and TNFα (e.g., TRAF2). These data thus parallel thedifferential effects of these ODC chimeric fusion polypeptides on TRAF6and TRAF2 polypeptide levels (FIG. 20).

We extended these studies of effects of the ODC/AZ system on endogenouspolypeptides to the tumor suppressor Rb. The Rb protein binds andsuppresses E2F-family transcription factors, thus preventing them fromactivating target genes (Chellappan et al., Cell 65:1053-1061, 1991).FIG. 23 shows the ablation of endogenous Rb expression by using ODC/AZsystem. We expressed in cells an ODC chimeric fusion polypeptidecontaining a Rb-binding peptide from the E7 protein, alone or incombination with AZ (FIG. 23A). Analysis of the levels of endogenous Rbprotein by immunoblotting showed that the combination of ODC-E7p and AZinduced nearly complete disappearance of the Rb protein. In contrast, Rbprotein levels were unaffected either by expressing ODC-E7p without AZ,or by expressing ODC control polypeptide with or without AZ (FIG. 23B).Measurements of E2F activity by using reporter gene assays demonstrateda marked increase in cells coexpressing ODC-E7p and AZ-I (FIG. 23C),consistent with the observed loss of Rb polypeptide. We conclude,therefore, that the ODC/AZ system is capable of ablating the function ofendogenous target polypeptides in cells.

Previous studies have demonstrated that ODC is degraded by the 26Sproteasome through a ubiquitin-independent mechanism, whereby AZ bindinginduces exposure of the C terminus of ODC and accelerates itsdegradation by 50- to 100-fold. Normally, this pathway is induced inresponse to polyamines (spermine, spermidine, and putresine), whichtriggers AZ production, thus providing a negative feedback loop formaintaining appropriate intracellular levels of these molecules(reviewed in Coffino, Nat. Rev. Mol. Cell Biol. 2:188-194, 2001). Weexploited the ODC/AZ system for targeting degradation of selectedpolypeptides in cells. The ODC/AZ system affords the advantage over mostubiquitin-pathway-based strategies that posttranslational modificationof the target polypeptide (by ubiquitination) is not required, thusproviding a more direct means of delivering ligand/target complexes tothe proteasome for degradation. Indeed, compared with a variety ofubiquitin-pathway-based approaches examined, we found the ODC/AZ systemto be more effective at achieving degradation of a test set of twelvetarget polypeptides for which interacting polypeptides are known.Successful degradation was achieved in five of twelve test cases,suggesting that some polypeptides are recalcitrant to this targetingapproach. Multiple explanations could account for the intractability ofcertain polypeptide targets, including (i) insufficient affinityinteractions of the protein ligands with their cellular targetpolypeptides; (ii) dissociation of ligand and target during digestion ofthe ODC-ligand fusion by the proteasome, thus stripping the targetpolypeptide off; and (iii) impeded entry of the target polypeptide intothe pore of the proteasome because of rigid protein structure,necessitating protein unfolding. Thus, the tractability of specificpolypeptide targets to degradation by the ODC/AZ system must beempirically determined.

A potential concern with the ODC/AZ system is that expression ofODC-fusion polypeptides or AZ in cells may alter polyamine levels,leading to artifactual changes in cell growth, chromatin structure, orother cellular events. Measurement of cell-division rates for HeLa andHEK293T cells used for our experiments revealed no apparent effect ofODC-fusion polypeptides or AZ, suggesting that at least some types ofcells are not particularly sensitive to these manipulations. However,more subtle changes in cells overexpressing ODC fusion polypeptides andAZ conceivably may occur and therefore should be considered ininterpreting data derived from use of the ODC/AZ-based approach totargeted protein degradation. The suitability of this approach may alsobe dependent on the endogenous levels of AZ and AZ inhibitors inparticular types of primary cells or cell lines.

This basic system can be improved in a number of ways. For instance,mutant versions of ODC that lack enzymatic activity but which preserveproteasome-dependent degradation obviate untoward effects on polyaminesynthesis, particularly non-dimerizing mutants which cannot bindendogenous ODC. Similarly, production of complementary pairs of ODC andAZ mutants that bind each other but fail to interact with theirendogenous (wild-type) counterparts provide a means to avoid effects onpolyamine synthesis. It should be noted, however, that AZ may haveadditional cellular targets besides ODC, a possibility that must beconsidered, including cyclin D1 and Smad1, at least in certain type ofcells (Newman et al., J. Biol. Chem. 279:41504-41511, 2004; Lin et al.,BMC Cell Biol. 3:15, 2002). One possible advantage of the ability ofpolyamines to induce AZ expression is that it might be possible toforego transfection of AZ-encoding plasmid by adding polyaminesorpolyamine analogues to the cell cultures.

Results obtained with the ODC/AZ system and other previously reportedapproaches for inducing proteasome-dependent degradation of specificproteins (Stack et al., Nat. Biotechnol. 18:1298-1302, 2000; Lindsten etal., Nat. Biotechnol. 21:897-902, 2003; Zhou et al., Mol. Cell6:751-756, 2000; Liu et al., Biochem. Biophys. Res. Commun.313:1023-1029, 2004; Sakamoto et al., Proc. Natl. Acad. Sci. USA98:8554-8559, 2001; Su et al., Proc. Natl. Acad. Sci. USA100:12729-12734, 2003) should be interpreted with understanding that theparticular protein ligand that is chosen may have multiple cellulartargets, including unknown or unanticipated protein targets in additionto known interacting proteins intended for targeted degradation. Thus,phenotypes created by these targeted polypeptide-degradation methodscould potentially reflect the loss of expression of several interactingpolypeptides. When searching for functions of gene products where morespecific methods such as antisense or small interfering RNA have failedto yield phenotypes, ablating the expression of the interrogated geneproduct's interacting partners may provide clues for eventuallyunderstanding its function. Moreover, targeted protein-degradationmethods that attack interacting proteins afford an approach for dealingwith multigene families, where a particular protein ligand may interactwith multiple members of a family of homologous gene products, therebyablating expression simultaneously of several redundant members andrevealing phenotypes that would be undetected by nucleic-acid-basedmethods for silencing gene expression at the mRNA.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

SUMMARY OF SEQUENCES

SEQ ID NO: 1 is a cDNA (and the deduced amino acid sequence) encoding aSiah 1α of the present invention.

SEQ ID NO:2 is the deduced amino acid sequence of a Siah 1α protein ofthe present invention encoded by SEQ ID NO: 1.

SEQ ID NO:3 is a cDNA (and the deduced amino acid sequence) encoding ahuman SIP-L polypeptide of the present invention.

SEQ ID NO:4 is the deduced amino acid sequence of a human SIP-L proteinof the present invention encoded by SEQ ID NO:3.

SEQ ID NO:5 is a cDNA (and the deduced amino acid sequence) encoding ahuman SIP-S polypeptide of the present invention.

SEQ ID NO:6 is the deduced amino acid sequence of a human SIP-S proteinof the present invention encoded by SEQ ID NO:5.

SEQ ID NO:7 is a cDNA (and the deduced amino acid sequence) encoding ahuman SAF-1α polypeptide of the present invention.

SEQ ID NO:8 is the deduced amino acid sequence of a SAF-1α protein ofthe present invention encoded by SEQ ID NO:7.

SEQ ID NO:9 is a cDNA (and the deduced amino acid sequence) encoding ahuman SAF-1β polypeptide of the present invention.

SEQ ID NO:10 is the deduced amino acid sequence of a SAF-1β proteinencoded by SEQ ID NO:9.

SEQ ID NO: 11 is a cDNA (and the deduced amino acid sequence) encoding ahuman SAF-2 polypeptide of the present invention.

SEQ ID NO: 12 is the deduced amino acid sequence of a SAF-2 proteinencoded by SEQ ID NO: 11.

SEQ ID NO: 13 is a cDNA (and the deduced amino acid sequence) encoding ahuman SAD polypeptide of the present invention.

SEQ ID NO: 14 is the deduced amino acid sequence of a SAD proteinencoded by SEQ ID NO: 13.

SEQ ID NO:50 is the amino acid sequence of the ODC-fusion protein.

1. An isolated chimeric protein that comprises: a target-protein bindingdomain operatively linked to a protein-degradation binding domain ofornithine decarboxylase, and a flexible linker sequence[Gly-Gly-Gly-Gly-Ser]₃ (SEQ ID NO: 61) between the target-proteinbinding domain and the protein-degradation binding domain of ornithinedecarboxylase, wherein binding of the target-protein binding domain ofthe chimeric protein to a target protein in a cell induces degradationof the target protein in the cell.
 2. The isolated chimeric protein ofclaim 1, wherein the protein-degradation binding domain of ornithinedecarboxylase is an ornithine decarboxylase C-terminal regionpolypeptide.
 3. The isolated chimeric protein of claim 1, comprising aprotein-degradation binding domain of human ornithine decarboxylase. 4.The isolated chimeric protein of claim 1, wherein theprotein-degradation binding domain of ornithine decarboxylase is a humanornithine decarboxylase C-terminal region polypeptide.
 5. The isolatedchimeric protein of claim 1, comprising, in order from N-terminus toC-terminus, (a) the target-protein binding domain, (b) the flexiblelinker sequence, and (c) the protein-degradation binding domain ofornithine decarboxylase.
 6. The isolated chimeric protein of claim 1,further comprising an epitope tag sequence.
 7. A pharmaceuticalcomposition comprising a therapeutically effective amount of thechimeric protein of claim 1, and a pharmaceutically acceptable carrier.8. The pharmaceutical composition of claim 7, further comprising apolyamine.